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US20210199644A1 - Treatment methods - Google Patents

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Publication number
US20210199644A1
US20210199644A1 US17/085,027 US202017085027A US2021199644A1 US 20210199644 A1 US20210199644 A1 US 20210199644A1 US 202017085027 A US202017085027 A US 202017085027A US 2021199644 A1 US2021199644 A1 US 2021199644A1
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US
United States
Prior art keywords
cancer
subject
tumor
level
antigens
Prior art date
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Application number
US17/085,027
Inventor
Jessica Baker Flechtner
Marie Lossky-Elias
Pamela M. Carroll
Hubert Lam
Lisa K. McNeil
Wendy Jane Broom
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ichor Medical Systems Inc
Original Assignee
Genocea Biosciences Inc
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Publication date
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Priority to US17/085,027 priority Critical patent/US20210199644A1/en
Assigned to GENOCEA BIOSCIENCES, INC. reassignment GENOCEA BIOSCIENCES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BROOM, Wendy Jane, CARROLL, PAMELA M., FLECHTNER, JESSICA BAKER, LAM, Hubert, LOSSKY-ELIAS, Marie, MCNEIL, Lisa K.
Publication of US20210199644A1 publication Critical patent/US20210199644A1/en
Assigned to ICHOR MEDICAL SYSTEMS, INC. reassignment ICHOR MEDICAL SYSTEMS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GENOCEA BIOSCIENCES, INC.
Abandoned legal-status Critical Current

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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A90/00—Technologies having an indirect contribution to adaptation to climate change
    • Y02A90/10—Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

Definitions

  • Cancer is characterized by proliferation of abnormal cells. Many treatments include costly and painful surgeries and chemotherapies. Although there is a growing interest in cancer therapies that target cancerous cells using a patient's own immune system, such therapies have had limited success.
  • the present invention features, inter alia, methods of identifying tumor antigens and potential tumor antigens of human lymphocytes, methods of selecting tumor antigens and potential tumor antigens, as well as compositions including the tumor antigens and potential tumor antigens, methods of making such compositions, and methods of using the tumor antigens and potential tumor antigens.
  • the invention also features methods of evaluating an immune response in a cancer subject, e.g., for identifying and/or selecting a cancer subject for initiation, continuation, modification, and/or discontinuation of a cancer therapy
  • the disclosure features a method of obtaining or generating a subject response profile.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads of the library with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression
  • APCs antigen
  • the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of obtaining or generating a target response profile.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads of the library with antigen presenting cells (APCs) from a subject who exhibits or previously exhibited a response to cancer, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased
  • the subject exhibits or previously exhibited at least one beneficial response to cancer.
  • the beneficial response comprises a positive clinical response, e.g., one or more positive clinical endpoints, to a cancer therapy or combination of therapies.
  • the beneficial response comprises a spontaneous response to a cancer.
  • the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer.
  • the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years).
  • the beneficial response comprises a positive cancer prognosis.
  • the beneficial response comprises a lack of measurable toxic responses or side effects to a cancer therapy or combination of therapies.
  • the subject exhibits or previously exhibited at least one deleterious or non-beneficial response to cancer.
  • the deleterious response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies.
  • the deleterious response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer.
  • the deleterious response comprises at least one relapse, recurrence, and/or metastasis of a cancer.
  • the deleterious response comprises a negative cancer prognosis.
  • the deleterious response comprises one or more toxic responses or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • the library used to obtain the target response profile is the same library used to obtain a subject response profile.
  • the method further comprises the step of repeating steps a) through e) with antigen presenting cells (APCs) and/or lymphocytes from additional subjects, to obtain a population-based or composite target response profile.
  • APCs antigen presenting cells
  • the target response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of identifying a subject as a candidate for cancer therapy.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secret
  • APCs anti
  • the method further comprises generating the target response profile by a method comprising: g) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; h) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; i) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and j) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain
  • the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer.
  • the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies.
  • the beneficial response comprises a spontaneous response to a cancer.
  • the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer.
  • the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years).
  • the beneficial response comprises a positive cancer prognosis.
  • the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer.
  • the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer.
  • the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer.
  • the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises selecting the candidate subject for initiation of a cancer therapy or combination of cancer therapies. In some embodiments, the method further comprises selecting the candidate subject for continuation of a cancer therapy or combination of cancer therapies. In some embodiments, the method comprises selecting the subject as a candidate subject (i) if the subject response profile is similar to the target response profile from a target subject who exhibits or previously exhibited one or more beneficial responses to the cancer therapy or combination, and/or (ii) if the subject response profile is dissimilar to the target response profile from a target subject who exhibits or previously exhibited one or more deleterious responses to the cancer therapy or combination. In some embodiments, the method further comprises administering the cancer therapy or combination of cancer therapies to the candidate subject.
  • the method further comprises selecting the candidate subject for modification of a cancer therapy. In some embodiments, the method further comprises selecting the candidate subject for discontinuation or non-initiation of a cancer therapy. In some embodiments, the method further comprises selecting the subject as a candidate subject for modification, discontinuation, and/or non-initiation of a cancer therapy (i) if the subject response profile is similar to the target response profile from a target subject who exhibits or previously exhibited one or more deleterious responses to the cancer therapy, and/or (ii) if the subject response profile is dissimilar to the target response profile from a target subject who exhibits or previously exhibited one or more beneficial responses to the cancer therapy. In some embodiments, the method further comprises modifying the cancer therapy administered to the candidate subject. In some embodiments, the method further comprises discontinuing or not initiating the cancer therapy to the candidate subject.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of selecting tumor antigens.
  • the, method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level) e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one
  • APCs anti
  • the method further comprises generating the target response profile by a method comprising: h) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; i) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; j) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and k) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain
  • the target response profile is from one or more target subjects who exhibit or previously exhibited one or more beneficial response to cancer.
  • the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies.
  • the beneficial response comprises a spontaneous response to a cancer.
  • the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer.
  • the beneficial response comprises a relapse, recurrence, and/or metastasis of a cancer e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years).
  • the beneficial response comprises a positive cancer prognosis.
  • the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious or non-beneficial response to cancer.
  • the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer.
  • the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer.
  • the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises selecting (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with a beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with deleterious or not beneficial responses to cancer.
  • the method further comprises administering to the subject an immunogenic composition comprising one or more of the selected antigens or immunogenic fragments thereof.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the method further comprises selecting (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with deleterious or not beneficial responses to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with beneficial responses to cancer.
  • the method further comprises administering to the subject an immunogenic composition that does not comprise one or more of the selected antigens or immunogenic fragments thereof.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secret
  • APCs anti
  • the method further comprises generating the target response profile by a method comprising: i) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; j) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; k) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and l) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain
  • the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer.
  • the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies.
  • the beneficial response comprises a spontaneous response to a cancer.
  • the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer.
  • the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years).
  • the beneficial response comprises a positive cancer prognosis.
  • the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises selecting and administering to the subject (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with one or more beneficial responses to cancer, and/or (i) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with one or more deleterious or not beneficial responses to cancer.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secreti
  • APCs anti
  • the method further comprises generating the target response profile by a method comprising: i) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; j) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; k) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and l) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain
  • the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer.
  • the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer.
  • the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer.
  • the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises selecting one or more tumor antigens that increase expression or secretion of immune mediators associated with deleterious or not beneficial responses to cancer, and/or one or more tumor antigens that inhibit and/or suppress expression or secretion of immune mediators associated with beneficial responses to cancer.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of selecting tumor antigens.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by one or more tumor antigens presented by one or more APCs by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor
  • APCs anti
  • the method further comprises comparing the subject response profile to a target response profile, e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison.
  • a target response profile e.g., a target response profile generated using a method described herein
  • the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer.
  • the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies.
  • the beneficial response comprises a spontaneous response to a cancer.
  • the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer.
  • the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years).
  • the beneficial response comprises a positive cancer prognosis.
  • the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises administering to the subject an immunogenic composition comprising one or more of the selected antigens or immunogenic fragments thereof.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of selecting tumor antigens.
  • the method comprises a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by one or more tumor antigens presented by one or more APCs by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens.
  • the method further comprises comparing the subject response profile to a target response profile, e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison.
  • a target response profile e.g., a target response profile generated using a method described herein
  • the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer.
  • the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer.
  • the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer.
  • the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises administering to the subject an immunogenic composition that does not comprise one or more of the selected antigens or immunogenic fragments thereof.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secreti
  • APCs anti
  • the method further comprises comparing the subject response profile to a target response profile, e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison, prior to administration of the immunogenic composition.
  • a target response profile e.g., a target response profile generated using a method described herein
  • the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer.
  • the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies.
  • the beneficial response comprises a spontaneous response to a cancer.
  • the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer.
  • the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years).
  • the beneficial response comprises a positive cancer prognosis.
  • the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control).
  • APCs antigen presenting cells
  • the method further comprises comparing the subject response profile to a target response profile e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison.
  • a target response profile e.g., a target response profile generated using a method described herein
  • the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer.
  • the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer.
  • the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer.
  • the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of identifying tumor antigens.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an enzyme, a sample
  • the disclosure features a method of selecting tumor antigens.
  • the method comprises: a) providing a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an enzyme, a sample
  • the method further comprises selecting from among the identified polypeptides one of more polypeptides that have a minimal effect on level of expression and/or secretion of one of more immune mediators.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of selecting potential tumor antigens.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level
  • the method further comprises repeating steps b) through e), or steps c) through e), with lymphocytes from the subject that have undergone one or more previous rounds of exposure to APCs.
  • the method further comprises selecting from among the identified tumor antigens (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial responses to cancer.
  • the method further comprises administering to the subject an immunogenic composition comprising one or more of the selected tumor antigens or selected polypeptides, or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject an immunogenic composition comprising a combination of one or more of the selected tumor antigens and selected polypeptides, or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of selecting tumor antigens.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level
  • the method further comprises administering to the subject an immunogenic composition that does not comprise one or more of the selected tumor antigens or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • LLO listeriolysin O
  • the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the disclosure features a method of inducing an immune response in a subject.
  • the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.
  • the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • the plurality of tumor antigens comprises at least 1, 3, 5, 10, 15, 20, 25, 30, 50, 100, 150, 250, 500, 750, 1000 or more different tumor antigens, or portions thereof; and/or determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens comprises measuring a level of one or more immune mediators; and/or the one or more immune mediators are selected from the group consisting of cytokines, soluble mediators, and cell surface markers expressed by the lymphocytes; and/or the one or more immune mediators are cytokines; and/or the one or more cytokines are selected from the group consisting of TRAIL, IFN-gamma, IL-12p70, IL-2, TNF-alpha, MIP1-alpha, MIP1-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10,
  • the disclosure features a method of inducing an immune response in a subject with one or more selected antigens, the method comprising: a) obtaining, providing or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a first subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the first subject, under conditions suitable for stimulation or inhibition and/or suppression of lymphocytes by a tumor antigen presented by one or more APCs; d) identifying one or more stimulatory tumor antigens that stimulate lymphocytes and identifying one or more non-stimulatory tumor antigens that do not stimulate lymphocytes, to produce a subject response profile; e) comparing the subject response profile to a target response profile, wherein the target response profile is from a
  • the method further comprises administering a cancer therapy to the subject.
  • the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • the disclosure features an immunogenic composition of the invention, comprising one or more antigens of the target response profile obtained or generated according to any of the methods described herein.
  • the disclosure features an immunogenic composition of the invention, comprising one or more antigens selected according to any of the methods described herein.
  • the disclosure features an immunogenic composition
  • an immunogenic composition comprising (i) one or more heparanase polypeptides or immunogenic fragments thereof and (ii) a SMAD4 polypeptide or immunogenic fragment thereof.
  • the one or more heparanase polypeptides or fragments and the SMAD4 polypeptide or fragment are each 8-29 amino acids in length.
  • the heparanase polypeptides comprise the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7.
  • the SMAD4 polypeptide comprises the amino acid sequence of SEQ ID NO:8.
  • the one or more immunogenic fragments consist of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total number of amino acids of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
  • the one or more immunogenic fragments consist of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 lacking about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids.
  • the one or more heparanse polypeptides comprise an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:6 or SEQ ID NO:7.
  • the SMAD4 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:8.
  • the disclosure features an immunogenic composition
  • a heparanase isoform 1 polypeptide or immunogenic fragment a heparanase isoform 2 polypeptide or immunogenic fragment
  • a SMAD4 polypeptide or immunogenic fragment a heparanase isoform 1 polypeptide or immunogenic fragment
  • a heparanase isoform 2 polypeptide or immunogenic fragment a heparanase isoform 2 polypeptide or immunogenic fragment
  • SMAD4 polypeptide or immunogenic fragment SMAD4 polypeptide or immunogenic fragment
  • the heparanase isoform 1 polypeptide or immunogenic fragment, the heparanase isoform 2 polypeptide or immunogenic fragment and the SMAD4 polypeptide or immunogenic fragment are each 8-29 amino acids in length.
  • the heparanase isoform 1 polypeptide comprises the amino acid sequence of SEQ ID NO:1
  • the heparanase isoform 2 polypeptide comprises the amino acid sequence of SEQ ID NO:2.
  • the SMAD4 polypeptide comprises the amino acid sequence of SEQ ID NO:3.
  • the one or more immunogenic fragments consist of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total number of amino acids of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, one or more immunogenic fragments consist of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 lacking about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids.
  • the heparanase isoform 1 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:6 and wherein the heparanase isoform 1 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:7.
  • the SMAD4 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:8.
  • the compositions further comprises an adjuvant.
  • methods of treating cancer comprises administering to a subject the immunogenic compositions described herein.
  • the subject has or is at risk of cancer, and/or exhibits one or more signs or symptoms of cancer, and/or exhibits one or more risk factors for cancer.
  • the cancer is colorectal cancer, melanoma, or lung cancer.
  • methods of inducing an immune response in a subject comprise administering to a subject the immunogenic compositions described herein.
  • the immune response comprises activation of one or more lymphocytes.
  • the one or more lymphocytes comprise CD4+ T cells.
  • the one or more lymphocytes comprise CD8+ T cells.
  • the one or more lymphocytes comprise NKT cells, gamma-delta T cells, or NK cells.
  • the one or more lymphocytes comprise any combination of CD4+ T cells, CD8+ T cells, NKT cells, gamma-delta T cells, and NK cells.
  • the immune response comprises an increased expression and/or secretion of one or more immune mediators relative to a control.
  • the lymphocyte signaling molecule is selected from among immune mediators.
  • the one or more immune mediators are cytokines.
  • the one or more cytokines are selected from TRAIL, IFN-gamma, IL-12p70, IL-2, TNF-alpha, MIP1-alpha, MIP1-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3-alpha, MCP1, and fractalkine.
  • the one or more immune mediators are soluble mediators. In some embodiments, the one or more soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin. In some embodiments, the one or more immune mediators are cell surface markers.
  • the one or more cell surface markers are selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD1 (CD279), FoxP3, TIGIT, CD160, BTLA, 2B4 (CD244), and KLRG1.
  • a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher than a control level.
  • a level of one or more expressed or secreted immune mediators that is at least one, two, or three standard deviations higher than the mean of a control level indicates lymphocyte activation.
  • a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) higher or lower than a median response level to a control indicates lymphocyte activation.
  • the immune response comprises a humoral response and/or a cellular response.
  • humoral response comprises an increase in magnitude of response or fold rise from baseline of antigen specific immunoglobulin G (IgG) levels and/or of antigen specific neutralizing antibody levels.
  • the humoral response comprises a 4-fold or greater rise in IgG titer from baseline.
  • the humoral response comprises a 2-fold or greater rise in 50% neutralizing antibody titer from baseline.
  • the cellular response comprises secretion of granzyme B (GrB).
  • the cellular response comprises an increase in magnitude of response or fold rise from baseline of granzyme B (GrB) levels.
  • the cellular response comprises an increase in IFN-gamma secretion for T cells.
  • the subject has or is at risk of cancer, and/or exhibits one or more signs or symptoms of cancer, and/or exhibits one or more risk factors for cancer.
  • the cancer is colorectal cancer, melanoma, or lung cancer.
  • the disclosure features a method for manufacturing an immunogenic composition, the method comprising combining one or more antigens identified by any method described herein and a carrier.
  • the antigen is produced using recombinant DNA technology in a suitable host cell.
  • the method comprises formulating the immunogenic composition as a pharmaceutical composition.
  • the disclosure features a method for manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: a. providing, preparing, or obtaining a plurality of antigenic compositions comprising a plurality of antigens, each composition comprising a different antigen; b. providing, preparing, or obtaining a target response profile, wherein the target response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; c.
  • the subject response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; d. comparing the target response profile to the subject response profile; e. selecting one or more antigens based on the comparison; and f. formulating at least a portion of one or more antigenic compositions comprising the one or more selected antigens as a pharmaceutical composition.
  • the selecting step comprises selecting one or more antigens that increase expression or secretion of immune mediators associated with a beneficial response to cancer, and/or one or more antigens that inhibit and/or suppress expression or secretion of immune mediators associated with deleterious or not beneficial responses to cancer.
  • the plurality of antigenic compositions are in solution, lyophilized, or on a synthetic matrix.
  • the disclosure features a method of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: preparing one or more antigens, or fragments thereof, identified by any of the method described herein; combining one or more antigens, or fragments thereof, wherein the one or more antigens or fragments thereof are selected according to the subject's response profile; and formulating the immunogenic composition as a pharmaceutical composition.
  • the disclosure features a method of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: preparing one or more antigens, or fragments thereof, identified by any method described herein; combining one or more antigens, or fragments thereof, wherein the one or more antigens or fragments thereof are selected according to whether or not the one or more antigens have been shown to stimulate, inhibit and/or suppress and/or have minimal effect on level of expression and/or secretion of one or more immune mediators by the subject's lymphocytes; and formulating the immunogenic composition as a pharmaceutical composition.
  • the disclosure features a method of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: preparing one or more antigens, or fragments thereof, identified by any method described herein;
  • the immunogenic composition as a pharmaceutical composition.
  • FIG. 1 is a graph showing IFN ⁇ secreted in supernatants by T cells from a representative melanoma patient who received immune checkpoint blockade therapy.
  • the T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor-associated antigens.
  • FIG. 2 is a graph showing the number of T cell antigens that stimulated cytokine secretion in supernatants by CD4 + T cells from melanoma patients who were non-responders or responders to immune checkpoint blockade therapy.
  • the T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor associated antigens.
  • FIG. 3 is a graph showing the number of T cell antigens that stimulated cytokine secretion in supernatants by CD8 + T cells from melanoma patients who were non-responders or responders to immune checkpoint blockade therapy.
  • the T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor-associated antigens.
  • FIG. 4 is a scatter plot showing good alignment between replicate measurements for cytokines secreted by T cells from a representative NSCLC patient after stimulation by autologous antigen presenting cells pulsed with E. coli expressing putative neoantigens.
  • FIG. 5 is a graph showing results for IFN ⁇ and TNF ⁇ secretion from CD8 + T cells from a representative NSCLC patient, collected pre- and post-checkpoint blockade therapy, after co-culture with autologous antigen presenting cells pulsed with E. coli expressing putative neoantigens.
  • FIG. 6 is a graph showing results for IFN ⁇ and TNF ⁇ secretion from CD4 + T cells from a representative NSCLC patient, collected pre- and post-checkpoint blockade therapy, after co-culture with autologous antigen presenting cells pulsed with E. coli expressing putative neoantigens.
  • FIG. 7 is a Venn diagram showing limited overlap between CD8 + -specific T cell neoantigens from a representative NSCLC patient, identified using methods of the disclosure and epitope prediction algorithms.
  • FIG. 8 is a schematic showing epitope predictions had a high false positive rate, missed relevant antigens and failed to identify suppressive and/or inhibitory neoantigens.
  • FIG. 9 is a graph showing IFN ⁇ and TNF- ⁇ secreted in supernatants by T cells from a representative patient with colorectal cancer.
  • the T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor-associated antigens.
  • NG neon green.
  • FIG. 10 is a graph showing the percentage of colorectal cancer patients who responded to each TAA, as measured by IFN ⁇ secretion that exceeded three standard deviations of the mean negative control response.
  • FIG. 11 is a graph showing results for IFN ⁇ and TNF- ⁇ secretion from CD8 + T cells from a patient with colorectal carcinoma after co-culture with antigen presenting cells pulsed with E. coli expressing 31 mutations unique to the patient.
  • FIGS. 12A and 12B are Venn diagrams representing the limited overlap between CD8 + -specific T cell neoantigens identified by ATLAS and epitope prediction algorithms.
  • FIG. 12A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ⁇ 1 for the mutant peptide but not for wild-type.
  • FIG. 12B represents epitopes predicted to have binding affinity below 500 nM or an IEDB percentile rank of ⁇ 1, irrespective of the wild-type counterpart predictions.
  • FIGS. 13A and 13B are Venn diagrams representing the limited overlap between CD8 + -specific T cell inhibitory and/or suppressive neoantigens identified by ATLAS and epitope prediction algorithms.
  • FIG. 13A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ⁇ 1 for the mutant peptide but not for wild-type.
  • FIG. 13B represents epitopes predicted to have binding affinity below 500 nM or an IEDB percentile rank of ⁇ 1, irrespective of the wild-type counterpart predictions.
  • FIG. 14 is a graph showing response profiles to 25 CRC-associated TAAs across CRC patients with all stages of disease using TNF- ⁇ and IFN- ⁇ secretion as an indicator for a recall response to a putative antigen.
  • FIGS. 15A and 15B are graphs showing the high frequency of T cell stimulatory responses to three novel ATLAS-identified TAAs in comparison to three TAAs that are or were in clinical development as a therapeutic vaccine.
  • FIG. 16 is a graph showing stimulatory response rates to 4 selected TAAs for both CD4 + and CD8 + T cell subsets from CRC patients with early or late stage disease and TNF- ⁇ and IFN- ⁇ cytokine release.
  • FIG. 17 is a graph showing normalized cytokine concentrations released in response to 4 selected TAAs in healthy individuals and donors with various disease states (polyps or CRC) for CD4 + and CD8 + T cell subsets and for TNF- ⁇ and IFN- ⁇ release.
  • FIG. 18 is a graph showing an exemplary empirical determination of T cell responses to profiled TAAs. Exemplary data is shown for a single lung cancer patient. T cell responses are reported as natural log concentrations extrapolated from the MSD standard curve and normalized to the patient's response to a negative control protein.
  • FIG. 19 is a graph showing shows frequent CD4 + T cell responses to two novel TAAs compared to previously described TAAs (NY-ESO-1, MUC1, and MAGEA3). Each point represents a patient's response to that TAA, normalized to the patient's response to a negative control protein. Stimulatory responses are colored black.
  • FIG. 20 is a graph showing CD4 + and CD8 + T cell responses to a broad range of TAAs from lung cancer patients.
  • FIG. 21 is a graph showing inhibitory and/or suppressive T cell responses detected in most profiled TAAs across lung cancer patients.
  • FIG. 22 is a graph showing a neoantigen screen with ATLAS identifying patient-specific CD4 + and CD8 + T cell responses. Each dot represents a technical replicate. Horizontal dotted lines indicate the cutoffs used to define stimulatory neoantigens and inhibitory and/or suppressive neoantigens at +3 and ⁇ 3 Median Absolute Deviations (MADs), respectively.
  • MADs Median Absolute Deviations
  • FIGS. 23A, 23B, 23C, and 23D show that algorithm prediction of MHC Class I binding does not accurately predict CD8 + T cell responses or types of response.
  • FIGS. 23A and 23C show the total numbers and overlap of neoantigens predicted by algorithm and observed in ATLAS.
  • FIGS. 24A and 24B are graphs showing that CD8 + T cell responses identified by ATLAS to candidate neoantigens are not enriched for any mutation type.
  • FIGS. 25A and 25B are graphs showing DNA mutant allele frequency is not associated with CD8 + T cell response frequency.
  • FIGS. 26A and 26B are graphs showing detection of a mutation in RNA does not predict whether the candidate neoantigen elicits a recall response in CD8 + T cells.
  • FIGS. 27A and 27B are graphs showing that CD8+ T cell responses identified by ATLAS to candidate neoantigens do not correlate with gene expression.
  • FIG. 28 is a graph illustrating the different cytokine response profiles elicited by 6 representative neoantigens in a screen of CD8 + T cells from a single patient.
  • FIG. 29 is a graph showing CD8+ T cell data for healthy donors and cancer patients.
  • IFN ⁇ secretion there was a large inhibitory response in the healthy donor cohort, that greatly exceeded the inhibitory responses in the cancer patient cohort. Conversely, there was a greater median inhibitory response in the cancer cohort when TNF ⁇ secretion was considered.
  • a peptide presented by an antigen presenting cell “activates” a lymphocyte if lymphocyte activity is detectably modulated after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur.
  • Any indicator of lymphocyte activity can be evaluated to determine whether a lymphocyte is activated, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers.
  • administration typically refers to the administration of a composition to a subject or system.
  • routes may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • administration may be systemic or local.
  • administration may be enteral or parenteral.
  • administration may be by injection (e.g., intramuscular, intravenous, or subcutaneous injection).
  • injection may involve bolus injection, drip, perfusion, or infusion.
  • administration may be topical.
  • administration may involve electro-osmosis, hemodialysis, infiltration, iontophoresis, irrigation, and/or occlusive dressing.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing.
  • Antigen refers to a molecule (e.g., a polypeptide) that elicits a specific immune response.
  • Antigen-specific immunological responses also known as adaptive immune responses, are mediated by lymphocytes (e.g., T cells, B cells, NK cells) that express antigen receptors (e.g., T cell receptors, B cell receptors).
  • an antigen is a T cell antigen, and elicits a cellular immune response.
  • an antigen is a B cell antigen, and elicits a humoral (i.e., antibody) response.
  • an antigen is both a T cell antigen and a B cell antigen.
  • the term “antigen” encompasses both a full-length polypeptide as well as a portion or immunogenic fragment of the polypeptide, and a peptide epitope within the polypeptides (e.g., a peptide epitope bound by a Major Histocompatibility Complex (MHC) molecule (e.g., MHC class I, or MHC class II)).
  • MHC Major Histocompatibility Complex
  • Antigen presenting cell refers to a cell that presents peptides on MHC class I and/or MHC class II molecules for recognition by T cells.
  • APC include both professional APC (e.g., dendritic cells, macrophages, B cells), which have the ability to stimulate na ⁇ ve lymphocytes, and non-professional APC (e.g., fibroblasts, epithelial cells, endothelial cells, glial cells).
  • APC are able to internalize (e.g., endocytose) members of a library (e.g., cells of a library of bacterial cells) that express heterologous polypeptides as candidate antigens.
  • Autolysin polypeptide is a polypeptide that facilitates or mediates autolysis of a cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell.
  • an autolysin polypeptide is a bacterial autolysin polypeptide.
  • Autolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in GenBank® under Acc. Nos. NP 388823.1, NP 266427.1, and POAGC3.1.
  • cancer refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they display an abnormally elevated proliferation rate and/or aberrant growth phenotype characterized by a significant loss of control of cell proliferation.
  • a cancer may be characterized by one or more tumors.
  • adrenocortical carcinoma astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, myeloid leukemia), lymphoma (e.g., Burkitt lymphoma [n ALL), acute myeloid leukemia (AML), chronic lymphocytic leuk
  • Cytolysin polypeptide is a polypeptide that has the ability to form pores in a membrane of a eukaryotic cell.
  • a cytolysin polypeptide when expressed in host cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell, facilitates release of host cell components (e.g., host cell macromolecules, such as host cell polypeptides) into the cytosol of the internalizing cell.
  • a cytolysin polypeptide is bacterial cytolysin polypeptide.
  • a cytolysin polypeptide is a cytoplasmic cytolysin polypeptide.
  • Cytolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in U.S. Pat. No. 6,004,815, and in GenBank® under Acc. Nos. NP_463733.1, NP_979614, NP_834769, YP_084586, YP_895748, YP_694620, YP_012823, NP_346351, YP_597752, BAB41212.2, NP_561079.1, YP_001198769, and NP_359331.1.
  • Cytoplasmic cytolysin polypeptide is a cytolysin polypeptide that has the ability to form pores in a membrane of a eukaryotic cell, and that is expressed as a cytoplasmic polypeptide in a bacterial cell.
  • a cytoplasmic cytolysin polypeptide is not significantly secreted by a bacterial cell.
  • Cytoplasmic cytolysin polypeptides can be provided by a variety of means. In some embodiments, a cytoplasmic cytolysin polypeptide is provided as a nucleic acid encoding the cytoplasmic ccytolysin polypeptide.
  • a cytoplasmic cytolysin polypeptide is provided attached to a bead.
  • a cytoplasmic cytolysin polypeptide has a sequence that is altered relative to the sequence of a secreted cytolysin polypeptide (e.g., altered by deletion or alteration of a signal sequence to render it nonfunctional).
  • a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a secretion-incompetent cell.
  • a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a cell that does not recognize and mediate secretion of a signal sequence linked to the cytolysin polypeptide.
  • a cytoplasmic cytolysin polypeptide is a bacterial cytolysin polypeptide.
  • heterologous refers to genes or polypeptides, refers to a gene or polypeptide that does not naturally occur in the organism in which it is present and/or being expressed, and/or that has been introduced into the organism by the hand of man.
  • a heterologous polypeptide is a tumor antigen described herein.
  • Immune mediator refers to any molecule that affects the cells and processes involved in immune responses. Immune mediators include cytokines, chemokines, soluble proteins, and cell surface markers.
  • an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
  • the effect of a particular agent or treatment may be direct or indirect.
  • an appropriate reference measurement may be or may comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
  • a peptide presented by an antigen presenting cell “stimulates” or is “stimulatory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control.
  • a peptide presented by an antigen presenting cell “suppresses”, “inhibits” or is “inhibitory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with deleterious or non-beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control.
  • an “invasin polypeptide” is a polypeptide that facilitates or mediates uptake of a cell (e.g., a bacterial cell) by a eukaryotic cell. Expression of an invasin polypeptide in a noninvasive bacterial cell confers on the cell the ability to enter a eukaryotic cell.
  • an invasin polypeptide is a bacterial invasin polypeptide.
  • an invasin polypeptide is a Yersinia invasin polypeptide (e.g., a Yersinia invasin polypeptide comprising a sequence disclosed in GenBank® under Acc. No. YP_070195.1).
  • Listeriolysin O refers to a listeriolysin O polypeptide of Listeria monocytogenes and truncated forms thereof that retain pore-forming ability (e.g., cytoplasmic forms of LLO, including truncated forms lacking a signal sequence).
  • an LLO is a cytoplasmic LLO. Exemplary LLO sequences are shown in Table 1, below.
  • Polypeptide generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate, however, that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) and immunogenic fragments of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity.
  • Primary cells refers to cells from an organism that have not been immortalized in vitro.
  • primary cells are cells taken directly from a subject (e.g., a human).
  • primary cells are progeny of cells taken from a subject (e.g., cells that have been passaged in vitro).
  • Primary cells include cells that have been stimulated to proliferate in culture.
  • a response refers to an alteration in a subject's condition that occurs as a result of, or correlates with, treatment.
  • a response is a beneficial response.
  • a beneficial response can include stabilization of a subject's condition (e.g., prevention or delay of deterioration expected or typically observed to occur absent the treatment), amelioration (e.g., reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or improvement in the prospects for cure of the condition, etc.
  • a beneficial response can include: the subject has a positive clinical response to cancer therapy or a combination of therapies; the subject has a spontaneous response to a cancer; the subject is in partial or complete remission from cancer; the subject has cleared a cancer; the subject has not had a relapse, recurrence or metastasis of a cancer; the subject has a positive cancer prognosis; the subject has not experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • the beneficial responses occurred in the past, or are ongoing.
  • a response is a deleterious or non-beneficial response.
  • a deleterious or non-beneficial response can include deterioration of a subject's condition, lack of amelioration (e.g., no reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or degradation in the prospects for cure of the condition, etc.
  • a deleterious or non-beneficial response can include: the subject has a negative clinical response to cancer therapy or a combination of therapies; the subject is not in remission from cancer; the subject has not cleared a cancer; the subject has had a relapse, recurrence or metastasis of a cancer; the subject has a negative cancer prognosis; the subject has experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • the deleterious or non-beneficial responses occurred in the past, or are ongoing.
  • a beneficial response in the context of a cell, organ, tissue, or cell component, e.g., a lymphocyte, “response”, “responsive”, or “responsiveness” refers to an alteration in cellular activity that occurs as a result of, or correlates with, administration of or exposure to an agent, e.g. a tumor antigen.
  • a beneficial response can include increased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject.
  • a beneficial response can include decreased expression and/or secretion of immune mediators associated with negative clinical response or outcomes in a subject.
  • a deleterious or non-beneficial response can include increased expression and/or secretion of immune mediators associated with negative clinical responses or outcomes in a subject. In certain embodiments, a deleterious or non-beneficial response can include decreased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject.
  • a response is a clinical response. In certain embodiments, a response is a cellular response. In certain embodiments, a response is a direct response. In certain embodiments, a response is an indirect response. In certain embodiments, “non-response”, “non-responsive”, or “non-responsiveness” mean minimal response or no detectable response.
  • a “minimal response” includes no detectable response.
  • presence, extent, and/or nature of response can be measured and/or characterized according to particular criteria.
  • criteria can include clinical criteria and/or objective criteria.
  • techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MM, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology.
  • a response of interest is a response of a tumor to a therapy
  • a response of interest is a response of a tumor to a therapy
  • methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:e143-52.
  • the exact response criteria can be selected in any appropriate manner, provided that when comparing groups of tumors, patients or experimental organism, and/or cells, organs, tissues, or cell components, the groups to be compared are assessed based on the same or comparable criteria for determining response rate.
  • One of ordinary skill in the art will be able to select appropriate criteria.
  • Tumor refers to an abnormal growth of cells or tissue.
  • a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • a tumor is associated with, or is a manifestation of, a cancer.
  • a tumor may be a disperse tumor or a liquid tumor.
  • a tumor may be a solid tumor.
  • Nivolumab and pembroluzimab have been approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for use in patients with advanced NSCLC who have previously been treated with chemotherapy. They have solidified the importance of T cell responses in control of tumors.
  • FDA Food and Drug Administration
  • EMA European Medicines Agency
  • Neoantigens potential cancer rejection antigens that are entirely absent from the normal human genome, are postulated to be relevant to tumor control; however, attempts to define them and their role in tumor clearance has been hindered by the paucity of available tools to define them in a biologically relevant and unbiased way (Schumacher and Schreiber, 2015 Science 348:69-74, Gilchuk et al., 2015 Curr Opin Immunol 34:43-51)
  • NSCLC non-small cell lung carcinoma
  • tumor antigens e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs)
  • TSAs tumor specific antigens
  • TAAs tumor associated antigens
  • CTAs cancer/testis antigens
  • tumor antigens includes both tumor antigens and potential tumor antigens.
  • methods of the present disclosure identified stimulatory tumor antigens that were not identified by known algorithms. Further, methods of the present disclosure identified suppressive and/or inhibitory tumor antigens that are not identifiable by known algorithms.
  • Methods of the present disclosure also identified polypeptides that are potential tumor antigens, i.e., polypeptides that activate T cells of non-cancerous subjects, but not T cells of subjects suffering from cancer.
  • the present disclosure also provides methods of selecting tumor antigens and potential tumor antigens, methods of using the selected tumor antigens and potential tumor antigens, immunogenic compositions comprising the selected tumor antigens and potential tumor antigens, and methods of manufacturing immunogenic compositions.
  • the present disclosure also provides methods of evaluating an immune response in a cancer subject, e.g., for identifying or selecting subjects for initiation, continuation, modification, and/or discontinuation of cancer therapy.
  • a library is a collection of members (e.g., cells or non-cellular particles, such as virus particles, liposomes, or beads (e.g., beads coated with polypeptides, such as in vitro translated polypeptides, e.g., affinity beads, e.g., antibody coated beads, or NTA-Ni beads bound to polypeptides of interest).
  • members of a library include (e.g., internally express or carry) polypeptides of interest described herein.
  • members of a library are cells that internally express polypeptides of interest described herein.
  • members of a library which are particles carry, and/or are bound to, polypeptides of interest.
  • a library is designed to be internalized by human antigen presenting cells so that peptides from library members, including peptides from internally expressed polypeptides of interest, are presented on MEW molecules of the antigen presenting cells for recognition by T cells.
  • Libraries can be used in assays that detect peptides presented by human MHC class I and MHC class II molecules.
  • Polypeptides expressed by the internalized library members are digested in intracellular endocytic compartments (e.g., phagosomes, endosomes, lysosomes) of the human cells and presented on MHC class II molecules, which are recognized by human CD4 + T cells.
  • library members include a cytolysin polypeptide, in addition to a polypeptide of interest.
  • library members include an invasin polypeptide, in addition to the polypeptide of interest.
  • library members include an autolysin polypeptide, in addition to the polypeptide of interest.
  • library members are provided with cells that express a cytolysin polypeptide (i.e., the cytolysin and polypeptide of interest are not expressed in the same cell, and an antigen presenting cell is exposed to members that include the cytolysin and members that include the polypeptide of interest, such that the antigen presenting cell internalizes both, and such that the cytolysin facilitates delivery of polypeptides of interest to the MHC class I pathway of the antigen presenting cell).
  • a cytolysin polypeptide can be constitutively expressed in a cell, or it can be under the control of an inducible expression system (e.g., an inducible promoter).
  • a cytolysin is expressed under the control of an inducible promoter to minimize cytotoxicity to the cell that expresses the cytolysin.
  • a cytolysin polypeptide perforates intracellular compartments in the human cell, allowing polypeptides expressed by the library members to gain access to the cytosol of the human cell.
  • Polypeptides released into the cytosol are presented on MHC class I molecules, which are recognized by CD8 + T cells.
  • a library can include any type of cell or particle that can be internalized by and deliver a polypeptide of interest (and a cytolysin polypeptide, in applications where a cytolysin polypeptide is desirable) to, antigen presenting cells for use in methods described herein.
  • a polypeptide of interest and a cytolysin polypeptide, in applications where a cytolysin polypeptide is desirable
  • antigen presenting cells for use in methods described herein.
  • the term “cell” is used throughout the present specification to refer to a library member, it is understood that, in some embodiments, the library member is a non-cellular particle, such as a virus particle, liposome, or bead.
  • members of the library include polynucleotides that encode the polypeptide of interest (and cytolysin polypeptide), and can be induced to express the polypeptide of interest (and cytolysin polypeptide) prior to, and/or during internalization by antigen presenting cells.
  • the cytolysin polypeptide is heterologous to the library cell in which it is expressed, and facilitates delivery of polypeptides expressed by the library cell into the cytosol of a human cell that has internalized the library cell.
  • Cytolysin polypeptides include bacterial cytolysin polypeptides, such as listeriolysin O (LLO), streptolysin O (SLO), and perfringolysin O (PFO). Additional cytolysin polypeptides are described in U.S. Pat. No. 6,004,815.
  • library members express LLO.
  • a cytolysin polypeptide is not significantly secreted by the library cell (e.g., less than 20%, 10%, 5%, or 1% of the cytolysin polypeptide produced by the cell is secreted).
  • the cytolysin polypeptide is a cytoplasmic cytolysin polypeptide, such as a cytoplasmic LLO polypeptide (e.g., a form of LLO which lacks the N-terminal signal sequence, as described in Higgins et al., Mol. Microbiol. 31(6):1631-1641,1999).
  • Exemplary cytolysin polypeptide sequences are shown in Table 1.
  • the listeriolysin O (43-25) sequence shown in the second row of Table 1 has a deletion of residues 3-25, relative to the LLO sequence in shown in the first row of Table 1, and is a cytoplasmic LLO polypeptide.
  • a cytolysin is expressed constitutively in a library host cell.
  • a cytolysin is expressed under the control of an inducible promoter. Cytolysin polypeptides can be expressed from the same vector, or from a different vector, as the polypeptide of interest in a library cell.
  • a library member (e.g., a library member which is a bacterial cell) includes an invasin that facilitates uptake by the antigen presenting cell.
  • a library member includes an autolysin that facilitates autolysis of the library member within the antigen presenting cell.
  • a library member includes both an invasin and an autolysin.
  • a library member which is an E. coli cell includes an invasin and/or an autolysin.
  • library cells that express an invasin and/or autolysin are used in methods that also employ non-professional antigen presenting cells or antigen presenting cells that are from cell lines. Isberg et al.
  • an autolysin has a feature that permits delayed lysis, e.g., the autolysin is temperature-sensitive or time-sensitive (see, e.g., Chang et al., 1995, 1 Bact.
  • cytolysins also include addiction (poison/antidote) autolysins, (see, e.g., Magnuson R, et al., 1996 , J Biol. Chem. 271(31), 18705-18710; Smith A S, et al., 1997 , Mol. Microbiol. 26(5), 961-970).
  • members of the library include bacterial cells.
  • the library includes non-pathogenic, non-virulent bacterial cells.
  • bacteria for use as library members include E. coli , mycobacteria, Listeria monocytogenes, Shigella flexneri, Bacillus subtilis , or Salmonella.
  • members of the library include eukaryotic cells (e.g., yeast cells). In some embodiments, members of the library include viruses (e.g., bacteriophages). In some embodiments, members of the library include liposomes. Methods for preparing liposomes that include a cytolysin and other agents are described in Kyung-Dall et al., U.S. Pat. No. 5,643,599. In some embodiments, members of the library include beads. Methods for preparing libraries comprised of beads are described, e.g., in Lam et al., Nature 354: 82-84, 1991, U.S. Pat. Nos. 5,510,240 and 7,262,269, and references cited therein.
  • a library is constructed by cloning polynucleotides encoding polypeptides of interest, or portions thereof, into vectors that express the polypeptides of interest in cells of the library.
  • the polynucleotides can be synthetically synthesized.
  • the polynucleotides can be cloned by designing primers that amplify the polynucleotides.
  • Primers can be designed using available software, such as Primer3Plus (available the following URL: bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; see Rozen and Skaletsky, In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp. 365-386, 2000). Other methods for designing primers are known to those of skill in the art. In some embodiments, primers are constructed so as to produce polypeptides that are truncated, and/or lack hydrophobic regions (e.g., signal sequences or transmembrane regions) to promote efficient expression.
  • hydrophobic regions e.g., signal sequences or transmembrane regions
  • the location of predicted signal sequences and predicted signal sequence cleavage sites in a given open reading frame (ORF) sequence can be determined using available software, see, e.g., Dyrl ⁇ v et al., J. Mol. Biol., 340:783-795, 2004, and the following URL: cbs.dtu.dk/services/SignalP/).
  • ORF open reading frame
  • Primers can also be designed to include sequences that facilitate subsequent cloning steps.
  • ORFs can be amplified directly from genomic DNA (e.g., genomic DNA of a tumor cell), or from polynucleotides produced by reverse transcription (RT-PCR) of mRNAs expressed by the tumor cell. RT-PCR of mRNA is useful, e.g., when the genomic sequence of interest contains intronic regions. PCR-amplified ORFs are cloned into an appropriate vector, and size, sequence, and expression of ORFs can be verified prior to use in immunological assays.
  • a polynucleotide encoding a polypeptide of interest is linked to a sequence encoding a tag (e.g., an N-terminal or C-terminal epitope tag) or a reporter protein (e.g., a fluorescent protein).
  • a tag e.g., an N-terminal or C-terminal epitope tag
  • a reporter protein e.g., a fluorescent protein.
  • Epitope tags and reporter proteins facilitate purification of expressed polypeptides, and can allow one to verify that a given polypeptide is properly expressed in a library host cell, e.g., prior to using the cell in a screen.
  • Useful epitope tags include, for example, a polyhistidine (His) tag, a V5 epitope tag from the P and V protein of paramyxovirus, a hemagglutinin (HA) tag, a myc tag, and others.
  • His polyhistidine
  • HA hemagglutinin
  • a polynucleotide encoding a polypeptide of interest is fused to a sequence encoding a tag which is a known antigenic epitope (e.g., an MHC class I- and/or MHC class II-restricted T cell epitope of a model antigen such as an ovalbumin), and which can be used to verify that a polypeptide of interest is expressed and that the polypeptide-tag fusion protein is processed and presented in antigen presentation assays.
  • a tag includes a T cell epitope of a murine T cell (e.g., a murine T cell line).
  • a polynucleotide encoding a polypeptide of interest is linked to a tag that facilitates purification and a tag that is a known antigenic epitope.
  • Useful reporter proteins include naturally occurring fluorescent proteins and their derivatives, for example, Green Fluorescent Protein ( Aequorea Victoria ) and Neon Green ( Branchiostoma lanceolatum ). Panels of synthetically derived fluorescent and chromogenic proteins are also available from commercial sources.
  • Polynucleotides encoding a polypeptide of interest are cloned into an expression vector for introduction into library host cells.
  • Various vector systems are available to facilitate cloning and manipulation of polynucleotides, such as the Gateway® Cloning system (Invitrogen).
  • expression vectors include elements that drive production of polypeptides of interest encoded by a polynucleotide in library host cells (e.g., promoter and other regulatory elements).
  • polypeptide expression is controlled by an inducible element (e.g., an inducible promoter, e.g., an IPTG- or arabinose-inducible promoter, or an IPTG-inducible phage T7 RNA polymerase system, a lactose (lac) promoter, a tryptophan (trp) promoter, a tac promoter, a trc promoter, a phage lambda promoter, an alkaline phosphatase (phoA) promoter, to give just a few examples; see Cantrell, Meth. in Mol. Biol., 235:257-276, Humana Press, Casali and Preston, Eds.).
  • an inducible element e.g., an inducible promoter, e.g., an IPTG- or arabinose-inducible promoter, or an IPTG-inducible phage T7 RNA polymerase system
  • lactose (lac) promoter e.g
  • polypeptides are expressed as cytoplasmic polypeptides.
  • the vector used for polypeptide expression is a vector that has a high copy number in a library host cell. In some embodiments, the vector used for expression has a copy number that is more than 25, 50, 75, 100, 150, 200, or 250 copies per cell. In some embodiments, the vector used for expression has a ColE1 origin of replication.
  • Useful vectors for polypeptide expression in bacteria include pET vectors (Novagen), Gateway® pDEST vectors (Invitrogen), pGEX vectors (Amersham Biosciences), pPRO vectors (BD Biosciences), pBAD vectors (Invitrogen), pLEX vectors (Invitrogen), pMALTM vectors (New England BioLabs), pGEMEX vectors (Promega), and pQE vectors (Qiagen).
  • Vector systems for producing phage libraries are known and include Novagen T7Select® vectors, and New England Biolabs Ph.D.TM Peptide Display Cloning System.
  • library host cells express (either constitutively, or when induced, depending on the selected expression system) a polypeptide of interest to at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total cellular protein.
  • the level a polypeptide available in or on a library member e.g., cell, virus particle, liposome, bead
  • a library member e.g., cell, virus particle, liposome, bead
  • antigen presenting cells exposed to a sufficient quantity of the library members are presented on MHC molecules polypeptide epitopes at a density that is comparable to the density presented by antigen presenting cells pulsed with purified peptides.
  • site-specific recombinases or rare-cutting restriction enzymes can be used to transfer polynucleotides between expression vectors in the proper orientation and reading frame (Walhout et al., Meth. Enzymol. 328:575-592, 2000; Marsischky et al., Genome Res. 14:2020-202, 2004; Blommel et al., Protein Expr. Purif. 47:562-570, 2006).
  • expressed polypeptides e.g., purified or partially purified polypeptides
  • liposomal membranes e.g., as described in Wassef et al., U.S. Pat. No. 4,863,874; Wheatley et al., U.S. Pat. No. 4,921,757; Huang et al., U.S. Pat. No. 4,925,661; or Martin et al., U.S. Pat. No. 5,225,212.
  • a library can be designed to include full length polypeptides and/or portions of polypeptides. Expression of full length polypeptides maximizes epitopes available for presentation by a human antigen presenting cell, thereby increasing the likelihood of identifying an antigen. However, in some embodiments, it is useful to express portions of polypeptides, or polypeptides that are otherwise altered, to achieve efficient expression.
  • polynucleotides encoding polypeptides that are large (e.g., greater than 1,000 amino acids), that have extended hydrophobic regions, signal peptides, transmembrane domains, or domains that cause cellular toxicity are modified (e.g., by C-terminal truncation, N-terminal truncation, or internal deletion) to reduce cytotoxicity and permit efficient expression a library cell, which in turn facilitates presentation of the encoded polypeptides on human cells.
  • Other types of modifications such as point mutations or codon optimization, may also be used to enhance expression.
  • a library can be designed to express polypeptides from at least 5%, 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of ORFs in a target cell (e.g., tumor cell).
  • a target cell e.g., tumor cell
  • a library expresses at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2500, 5000, 10,000, or more different polypeptides of interest, each of which may represent a polypeptide encoded by a single full length polynucleotide or portion thereof.
  • assays may focus on identifying antigens that are secreted polypeptides, cell surface-expressed polypeptides, or virulence determinants, e.g., to identify antigens that are likely to be targets of both humoral and cell mediated immune responses.
  • libraries can include tags or reporter proteins that allow one to easily purify, analyze, or evaluate MHC presentation, of the polypeptide of interest.
  • polypeptides expressed by a library include C-terminal tags that include both an MHC class I and an MHC class II-restricted T cell epitope from a model antigen, such as chicken ovalbumin (OVA).
  • OVA chicken ovalbumin
  • Library protein expression and MHC presentation is validated using these epitopes.
  • the epitopes are OVA 247-265 and OVA 258-265 respectfully, corresponding to positions in the amino acid sequence found in GenBank® under Acc. No. NP_990483.
  • T cell hybridomas e.g., B3Z T hybridoma cells, which are H2-K b restricted, and KZO T hybridoma cells, which are H2-A k restricted
  • T cell hybridomas e.g., B3Z T hybridoma cells, which are H2-K b restricted, and KZO T hybridoma cells, which are H2-A k restricted
  • Sets of library members e.g., bacterial cells
  • an array e.g., on a solid support, such as a 96-well plate
  • members in each location express a different polypeptide of interest, or a different set of polypeptides of interest.
  • library members also have utility in assays to identify B cell antigens.
  • lysate prepared from library members that include polypeptides of interest can be used to screen a sample comprising antibodies (e.g., a serum sample) from a subject (e.g., a subject who has been exposed to an infectious agent of interest, a subject who has cancer, and/or a control subject), to determine whether antibodies present in the subject react with the polypeptide of interest.
  • Suitable methods for evaluating antibody reactivity are known and include, e.g., ELISA assays.
  • methods and compositions described herein can be used to identify and/or detect immune responses to a polypeptide of interest.
  • a polypeptide of interest is encoded by an ORF from a target tumor cell, and members of a library include (e.g., internally express or carry) ORFs from a target tumor cell.
  • members of a library include (e.g., internally express or carry) ORFs from a target tumor cell.
  • a library can be used in methods described herein to assess immune responses to one or more polypeptides of interest encoded by one or more ORFs.
  • methods of the disclosure identify one or more polypeptides of interest as stimulatory antigens (e.g., that stimulate an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators).
  • methods of the disclosure identify one or more polypeptides of interest as antigens or potential antigens that have minimal or no effect on an immune response (e.g., expression and/or secretion of one or more immune mediators).
  • methods of the disclosure identify one or more polypeptides of interest as inhibitory and/or suppressive antigens (e.g., that inhibit, suppress, down-regulate, impair, and/or prevent an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators).
  • methods of the disclosure identify one or more polypeptides of interest as tumor antigens or potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs).
  • TSAs tumor specific antigens
  • TAAs tumor associated antigens
  • CTAs cancer/testis antigens
  • a polypeptide of interest is a putative tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more putative tumor antigens.
  • members of a library include (e.g., internally express or carry) putative tumor antigens (e.g., a polypeptide previously identified (e.g., by a third party) as a tumor antigen, e.g., identified as a tumor antigen using a method other than a method of the present disclosure).
  • a putative tumor antigen is a tumor antigen described herein.
  • such libraries can be used to assess whether and/or the extent to which such putative tumor antigen mediates an immune response.
  • methods of the disclosure identify one or more putative tumor antigens as stimulatory antigens. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as antigens that have minimal or no effect on an immune response. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as inhibitory and/or suppressive antigens.
  • a polypeptide of interest is a pre-selected tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more pre-selected tumor antigens.
  • members of a library include (e.g., internally express or carry) one or more polypeptides identified as tumor antigens using a method of the present disclosure and/or using a method other than a method of the present disclosure.
  • such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein.
  • methods of the disclosure identify one or more pre-selected tumor antigens as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
  • a polypeptide of interest is a known tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more known tumor antigens.
  • members of a library include (e.g., internally express or carry) one or more polypeptides identified as a tumor antigen using a method of the present disclosure and/or using a method other than a method of the present disclosure.
  • such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein.
  • methods of the disclosure identify one or more known tumor antigens as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
  • a polypeptide of interest is a potential tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more potential tumor antigens.
  • members of a library include (e.g., internally express or carry) one or more polypeptides identified as being of interest, e.g., encoding mutations associated with a tumor, using a method of the present disclosure and/or using a method other than a method of the present disclosure.
  • such libraries can be used to assess whether and/or the extent to which such polypeptides mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein.
  • methods of the disclosure identify one or more polypeptides as stimulatory antigens for one or more subjects.
  • methods of the disclosure identify one or more polypeptides as antigens that have minimal or no effect on an immune response for one or more subjects.
  • methods of the disclosure identify one or more polypeptides as inhibitory and/or suppressive antigens for one or more subjects.
  • Polypeptides of interest used in methods and systems described herein include tumor antigens amd potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), and/or cancer/testis antigens (CTAs).
  • TSAs tumor specific antigens
  • TAAs tumor associated antigens
  • CTAs cancer/testis antigens
  • Exemplary tumor antigens include, e.g., MART-1/MelanA (MART-I or MLANA), gp100 (Pmel 17 or SILV), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3 (also known as HIP8), BAGE, GAGE-1, GAGE-2, p15, Calcitonin, Calretinin, Carcinoembryonic antigen (CEA), Chromogranin, Cytokeratin, Desmin, Epithelial membrane protein (EMA), Factor VIII, Glial fibrillary acidic protein (GFAP), Gross cystic disease fluid protein (GCDFP-15), HMB-45, Human chorionic gonadotropin (hCG), inhibin, lymphocyte marker, MART-1 (Melan-A), Myo D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen
  • TSAs Tumor specific antigens
  • TSAs are tumor antigens that are not encoded in normal host genome (see, e.g., Yarchoan et al., Nat. Rev. Cancer. 2017 Feb. 24. doi: 10.1038/nrc.2016.154; Gubin et al., J. Clin. Invest. 125:3413-3421 (2015)).
  • TSAs arise from somatic mutations and/or other genetic alterations.
  • TSAs arise from missense or in-frame mutations.
  • TSAs arise from frame-shift mutations or loss-of-stop-codon mutations.
  • TSAs arise from insertion or deletion mutations.
  • TSAs arise from duplication or repeat expansion mutations. In some embodiments, TSAs arise from splice variants or improper splicing. In some embodiments, TSAs arise from gene fusions. In some embodiments, TSAs arise from translocations. In some embodiments, TSAs include oncogenic viral proteins. For example, as with Merkel cell carcinoma (MCC) associated with the Merkel cell polyomavirus (MCPyV) and cancers of the cervix, oropharynx and other sites associated with the human papillomavirus (HPV), TSAs include proteins encoded by viral open reading frames.
  • MCC Merkel cell carcinoma
  • MCPyV Merkel cell polyomavirus
  • HPV human papillomavirus
  • TSAs are specific (personal) to a subject.
  • TSAs are shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1-10%, or more of subjects suffering from a cancer.
  • TSAs shared by more than one subject may be known or pre-selected.
  • a TSA is encoded by an open reading frame from a virus.
  • a library can be designed to express polypeptides from one of the following viruses: an immunodeficiency virus (e.g., a human immunodeficiency virus (HIV), e.g., HIV-1, HIV-2), a hepatitis virus (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus, non-A and non-B hepatitis virus), a herpes virus (e.g., herpes simplex virus type I (HSV-1), HSV-2, Varicella-zoster virus, Epstein Barr virus, human cytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8), a poxvirus (e.g., variola, vaccinia, monkeypox, Molluscum contagiosum virus), an influenza virus, a human pap
  • HIV human immuno
  • Tumor specific antigens are known in the art, any of which can be used in methods described herein.
  • gene sequences encoding polypeptides that are potential or putative neoantigens are determined by sequencing the genome and/or exome of tumor tissue and healthy tissue from a subject having cancer using next generation sequencing technologies.
  • genes that are selected based on their frequency of mutation and ability to encode a potential or putative neoantigen are sequenced using next-generation sequencing technology. Next-generation sequencing applies to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChIP-sequencing), and epigenome characterization (de Magalhaes et al.
  • Next-generation sequencing can be used to rapidly reveal the presence of discrete mutations such as coding mutations in individual tumors, e.g., single amino acid changes (e.g., missense mutations, in-frame mutations) or novel stretches of amino acids generated by frame-shift insertions, deletions, gene fusions, read-through mutations in stop codons, duplication or repeat expansion mutations, and translation of splice variants or improperly spliced introns, and translocations (e.g., “neoORFs”).
  • single amino acid changes e.g., missense mutations, in-frame mutations
  • novel stretches of amino acids generated by frame-shift insertions, deletions, gene fusions, read-through mutations in stop codons, duplication or repeat expansion mutations e.g., “neoORFs”.
  • Another method for identifying potential or putative neoantigens is direct protein sequencing.
  • Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn) including tandem mass spectrometry (MS/MS)) can also be used to identify neoantigens.
  • MSn multidimensional MS techniques
  • MS/MS tandem mass spectrometry
  • Such proteomic approaches can be used for rapid, highly automated analysis (see, e.g., Gevaert et al., Electrophoresis 21:1145-1154 (2000)).
  • High-throughput methods for de novo sequencing of unknown proteins can also be used to analyze the proteome of a subject's tumor to identify expressed potential or putative neoantigens.
  • meta shotgun protein sequencing may be used to identify expressed potential or putative neoantigens (see e.g., Guthals et al. (2012) Molecular and Cellular Proteomics 11(10):1084-96).
  • Potential or putative neoantigens may also be identified using MHC multimers to identify neoantigen-specific T cell responses.
  • MHC tetramer-based screening techniques see e.g., Hombrink et al. (2011) PLoS One; 6(8): e22523; Hadrup et al. (2009) Nature Methods, 6(7):520-26; van Rooij et al. (2013) Journal of Clinical Oncology, 31:1-4; and Heemskerk et al. (2013) EMBO Journal, 32(2):194-203).
  • one or more known or pre-selected tumor specific antigens, or one or more potential or putative tumor specific antigens identified using one of these methods can be included in a library described herein.
  • Tumor associated antigens include proteins encoded in a normal genome (see, e.g., Ward et al., Adv. Immunol. 130:25-74 (2016)).
  • TAAs are either normal differentiation antigens or aberrantly expressed normal proteins.
  • WT1 Wilms tumor 1
  • Her2/neu Kawashima et al., Cancer Res. 59:431-435 (1999)
  • TAAs Post-translational modifications, such as phosphorylation, of proteins may also lead to formation of TAAs (Doyle, J. Biol. Chem. 281:32676-32683 (2006); Cobbold, Sci. Transl. Med. 5:203ra125 (2013)).
  • TAAs are generally shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1-10%, 1-20%, or more of subjects suffering from a cancer.
  • TAAs are known or pre-selected tumor antigens.
  • TAAs are potential or putative tumor antigens.
  • Cancer/testis antigens are expressed by various tumor types and by reproductive tissues (for example, testes, fetal ovaries and trophoblasts) but have limited or no detectable expression in other normal tissues in the adult and are generally not presented on normal reproductive cells, because these tissues do not express MHC class I molecules (see, e.g., Coulie et al., Nat. Rev. Cancer 14:135-146 (2014); Simpson et al., Nat. Rev. Cancer 5:615-625 (2005); Scanlan et al., Immunol. Rev. 188:22-32 (2002)).
  • the present invention provides, inter alia, compositions and methods for identifying tumor antigens recognized by human immune cells.
  • Human antigen presenting cells express ligands for antigen receptors and other immune activation molecules on human lymphocytes. Given differences in MHC peptide binding specificities and antigen processing enzymes between species, antigens processed and presented by human cells are more likely to be physiologically relevant human antigens in vivo than antigens identified in non-human systems. Accordingly, methods of identifying these antigens employ human cells to present candidate tumor antigen polypeptides. Any human cell that internalizes library members and presents polypeptides expressed by the library members on MHC molecules can be used as an antigen presenting cell according to the present disclosure. In some embodiments, human cells used for antigen presentation are primary human cells.
  • the cells can include peripheral blood mononuclear cells (PBMC) of a human.
  • peripheral blood cells are separated into subsets (e.g., subsets comprising dendritic cells, macrophages, monocytes, B cells, or combinations thereof) prior to use in an antigen presentation assay.
  • a subset of cells that expresses MHC class II is selected from peripheral blood.
  • a cell population including dendritic cells is isolated from peripheral blood.
  • a subset of dendritic cells is isolated (e.g., plasmacytoid, myeloid, or a subset thereof).
  • Human dendritic cell markers include CD1c, CD1a, CD303, CD304, CD141, and CD209. Cells can be selected based on expression of one or more of these markers (e.g., cells that express CD303, CD1c, and CD141).
  • Dendritic cells can be isolated by positive selection from peripheral blood using commercially available kits (e.g., from Miltenyi Biotec Inc.). In some embodiments, the dendritic cells are expanded ex vivo prior to use in an assay. Dendritic cells can also be produced by culturing peripheral blood cells under conditions that promote differentiation of monocyte precursors into dendritic cells in vitro. These conditions typically include culturing the cells in the presence of cytokines such as GM-CSF and IL-4 (see, e.g., Inaba et al., Isolation of dendritic cells, Curr. Protoc. Immunol . May; Chapter 3: Unit 3.7, 2001).
  • cytokines such as GM-CSF and IL-4
  • CD34 + hematopoietic stem and progenitor cells are isolated from peripheral blood or bone marrow and expanded in vitro in culture conditions that include one or more of Flt3-L, IL-1, IL-3, and c-kit ligand.
  • immortalized cells that express human MHC molecules are used for antigen presentation.
  • assays can employ COS cells transfected with human MHC molecules or HeLa cells.
  • both the antigen presenting cells and immune cells used in the method are derived from the same subject (e.g., autologous T cells and APC are used).
  • DC dendritic cells
  • DC are used with T- and DC-depleted cells in an assay, at a ratio of 1:2, 1:3, 1:4, or 1:5.
  • the antigen presenting cells and immune cells used in the method are derived from different subjects (e.g., heterologous T cells and APC are used).
  • Antigen presenting cells can be isolated from sources other than peripheral blood.
  • antigen presenting cells can be taken from a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, lymph nodes, spleen, bone marrow, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, or other tissue, for use in screening assays.
  • cells are taken from a tissue that is the site of an active immune response (e.g., an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
  • Antigen presenting cells useful in methods described herein are not limited to “professional” antigen presenting cells.
  • non-professional antigen presenting cells can be utilized effectively in the practice of methods of the present disclosure.
  • Non-professional antigen presenting cells include fibroblasts, epithelial cells, endothelial cells, neuronal/glial cells, lymphoid or myeloid cells that are not professional antigen presenting cells (e.g., T cells, neutrophils), muscle cells, liver cells, and other types of cells.
  • Antigen presenting cells are cultured with library members that express a polypeptide of interest (and, if desired, a cytolysin polypeptide) under conditions in which the antigen presenting cells internalize, process and present polypeptides expressed by the library members on WIC molecules.
  • library members are killed or inactivated prior to culture with the antigen presenting cells.
  • Cells or viruses can be inactivated by any appropriate agent (e.g., fixation with organic solvents, irradiation, freezing).
  • the library members are cells that express ORFs linked to a tag (e.g., a tag which comprises one or more known T cell epitopes) or reporter protein, expression of which has been verified prior to the culturing.
  • antigen presenting cells are incubated with library members at 37° C. for between 30 minutes and 5 hours (e.g., for 45 min. to 1.5 hours). After the incubation, the antigen presenting cells can be washed to remove library members that have not been internalized. In certain embodiments, the antigen presenting cells are non-adherent, and washing requires centrifugation of the cells. The washed antigen presenting cells can be incubated at 37° C. for an additional period of time (e.g., 30 min. to 2 hours) prior to exposure to lymphocytes, to allow antigen processing. In some embodiments, it is desirable to fix and kill the antigen presenting cells prior to exposure to lymphocytes (e.g., by treating the cells with 1% paraformaldehyde).
  • antigen presenting cell and library member numbers can be varied, so long as the library members provide quantities of polypeptides of interest sufficient for presentation on WIC molecules.
  • antigen presenting cells are provided in an array, and are contacted with sets of library cells, each set expressing a different polypeptide of interest.
  • each location in the array includes 1 ⁇ 10 3 -1 ⁇ 10 6 antigen presenting cells, and the cells are contacted with 1 ⁇ 10 3 -1 ⁇ 10 8 library cells which are bacterial cells.
  • antigen presenting cells can be freshly isolated, maintained in culture, and/or thawed from frozen storage prior to incubation with library cells, or after incubation with library cells.
  • human lymphocytes are tested for antigen-specific reactivity to antigen presenting cells, e.g., antigen presenting cells that have been incubated with libraries expressing polypeptides of interest as described above.
  • the methods of the present disclosure permit rapid identification of human antigens using pools of lymphocytes isolated from an individual, or progeny of the cells. The detection of antigen-specific responses does not rely on laborious procedures to isolate individual T cell clones.
  • the human lymphocytes are primary lymphocytes.
  • human lymphocytes are NKT cells, gamma-delta T cells, or NK cells.
  • a population of lymphocytes having a specific marker or other feature can be used.
  • a population of T lymphocytes is isolated.
  • a population of CD4 + T cells is isolated.
  • a population of CD8 + T cells is isolated.
  • CD8 + T cells recognize peptide antigens presented in the context of MHC class I molecules.
  • the CD8 + T cells are used with antigen presenting cells that have been exposed to library host cells that co-express a cytolysin polypeptide, in addition to a polypeptide of interest.
  • T cell subsets that express other cell surface markers may also be isolated, e.g., to provide cells having a particular phenotype. These include CLA (for skin-homing T cells), CD25, CD30, CD69, CD154 (for activated T cells), CD45RO (for memory T cells), CD294 (for Th2 cells), ⁇ / ⁇ TCR-expressing cells, CD3 and CD56 (for NK T cells). Other subsets can also be selected.
  • Lymphocytes can be isolated, and separated, by any means known in the art (e.g., using antibody-based methods such as those that employ magnetic bead separation, panning, or flow cytometry). Reagents to identify and isolate human lymphocytes and subsets thereof are well known and commercially available.
  • Lymphocytes for use in methods described herein can be isolated from peripheral blood mononuclear cells, or from other tissues in a human.
  • lymphocytes are taken from tumors, lymph nodes, a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, spleen, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, peritoneal cavity, bone marrow, or other tissues.
  • cells are taken from a tissue that is the site of an active immune response (e.g., an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
  • Lymphocytes taken from an individual can be maintained in culture or frozen until use in antigen presentation assays.
  • freshly isolated lymphocytes can be stimulated in vitro by antigen presenting cells exposed to library cells as described above.
  • these lymphocytes exhibit detectable stimulation without the need for prior non-antigen specific expansion.
  • primary lymphocytes also elicit detectable antigen-specific responses when first stimulated non-specifically in vitro.
  • lymphocytes are stimulated to proliferate in vitro in a non-antigen specific manner, prior to use in an antigen presentation assay.
  • Lymphocytes can also be stimulated in an antigen-specific manner prior to use in an antigen presentation assay.
  • cells are stimulated to proliferate by a library (e.g., prior to use in an antigen presentation assay that employs the library). Expanding cells in vitro provides greater numbers of cells for use in assays.
  • Primary T cells can be stimulated to expand, e.g., by exposure to a polyclonal T cell mitogen, such as phytohemagglutinin or concanavalin, by treatment with antibodies that stimulate proliferation, or by treatment with particles coated with the antibodies.
  • T cells are expanded by treatment with anti-CD2, anti-CD3, and anti-CD28 antibodies.
  • T cells are expanded by treatment with interleukin-2.
  • lymphocytes are thawed from frozen storage and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells.
  • lymphocytes are thawed from frozen storage and are not expanded prior to contacting with antigen presenting cells.
  • lymphocytes are freshly isolated and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells.
  • T cells are cultured with antigen presenting cells prepared according to the methods described above, under conditions that permit T cell recognition of peptides presented by MHC molecules on the antigen presenting cells.
  • T cells are incubated with antigen presenting cells at 37° C. for between 12-48 hours (e.g., for 24 hours).
  • T cells are incubated with antigen presenting cells at 37° C. for 3, 4, 5, 6, 7, or 8 days. Numbers of antigen presenting cells and T cells can be varied.
  • the ratio of T cells to antigen presenting cells in a given assay is 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 20:1, 25:1, 30:1, 32:1, 35:1 or 40:1.
  • antigen presenting cells are provided in an array (e.g., in a 96-well plate), wherein cells in each location of the array have been contacted with sets of library cells, each set including a different polypeptide of interest.
  • each location in the array includes 1 ⁇ 10 3 -1 ⁇ 10 6 antigen presenting cells, and the cells are contacted with 1 ⁇ 10 3 -1 ⁇ 10 6 T cells.
  • Lymphocyte activation can be detected by any means known in the art, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers.
  • culture supernatants are harvested and assayed for increased and/or decreased expression and/or secretion of one or more polypeptides associated with activation, e.g., a cytokine, soluble mediator, cell surface marker, or other immune mediator.
  • the one or more cytokines are selected from TRAIL, IFN-gamma, IL-12p′70, IL-2, TNF-alpha, MIP1-alpha, MIP1-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3-alpha, and fractalkine.
  • the one or more soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin.
  • the one or more cell surface markers are selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD1 (CD279), FoxP3, TIGIT, CD160, BTLA, 2B4 (CD244), and KLRG1.
  • Cytokine secretion in culture supernatants can be detected, e.g., by ELISA, bead array, e.g., with a Luminex® analyzer. Cytokine production can also be assayed by RT-PCR of mRNA isolated from the T cells, or by ELISPOT analysis of cytokines released by the T cells.
  • proliferation of T cells in the cultures is determined (e.g., by detecting 3 H thymidine incorporation).
  • target cell lysis is determined (e.g., by detecting T cell dependent lysis of antigen presenting cells labeled with Na 2 51 CrO 4 ). Target cell lysis assays are typically performed with CD8 + T cells.
  • Protocols for these detection methods are known. See, e.g., Current Protocols In Immunology , John E. Coligan et al. (eds), Wiley and Sons, New York, N.Y., 2007.
  • appropriate controls are used in these detection methods, e.g., to adjust for non-antigen specific background activation, to confirm the presenting capacity of antigen presenting cells, and to confirm the viability of lymphocytes.
  • antigen presenting cells and lymphocytes used in the method are from the same individual. In some embodiments, antigen presenting cells and lymphocytes used in the method are from different individuals.
  • antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, e.g., to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g., to enhance detection of responses, or to enhance weak initial responses.
  • antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, and antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g., to enhance detection of responses, or to enhance weak initial responses.
  • antigen presentation assays are repeated using antigen presenting cells and lymphocytes from different individuals, e.g., to identify antigens recognized by multiple individuals, or compare reactivities that differ between individuals.
  • Humans that have cancer may have lymphocytes that specifically recognize tumor antigens, which are the product of an adaptive immune response arising from prior exposure. In some embodiments, these cells are present at a higher frequency than cells from an individual who does not have cancer, and/or the cells are readily reactivated when re-exposed to the proper antigenic stimulus (e.g., the cells are “memory” cells). Thus, humans that have or have had cancer are particularly useful donors of cells for identifying antigens in vitro. The individual may be one who has recovered from cancer.
  • the individual has been recently diagnosed with cancer (e.g., the individual was diagnosed less than one year, three months, two months, one month, or two weeks, prior to isolation of lymphocytes and/or antigen presenting cells from the individual). In some embodiments, the individual was first diagnosed with cancer more than three months, six months, or one year prior to isolation of lymphocytes and/or antigen presenting cells.
  • lymphocytes are screened against antigen presenting cells that have been contacted with a library of cells whose members express or carry polypeptides of interest, and the lymphocytes are from an individual who has not been diagnosed with cancer. In some embodiments, such lymphocytes are used to determine background (i.e., non-antigen-specific) reactivities. In some embodiments, such lymphocytes are used to identify antigens, reactivity to which exists in non-cancer individuals.
  • Cells from multiple donors can be collected and assayed in methods described herein.
  • cells from multiple donors are assayed in order to determine if a given tumor antigen is reactive in a broad portion of the population, or to identify multiple tumor antigens that can be later combined to produce an immunogenic composition that will be effective in a broad portion of the population.
  • Antigen presentation assays are useful in the context of both infectious and non-infectious diseases.
  • the methods described herein are applicable to any context in which a rapid evaluation of human cellular immunity is beneficial.
  • antigenic reactivity to polypeptides that are differentially expressed by neoplastic cells is evaluated.
  • Sets of nucleic acids differentially expressed by neoplastic cells have been identified using established techniques such as subtractive hybridization.
  • Methods described herein can be used to identify antigens that were functional in a subject in which an anti-tumor immune response occurred.
  • methods are used to evaluate whether a subject has lymphocytes that react to a tumor antigen or set of tumor antigens.
  • antigen presentation assays are used to examine reactivity to autoantigens in cells of an individual, e.g., an individual predisposed to, or suffering from, an autoimmune condition. Such methods can be used to provide diagnostic or prognostic indicators of the individual's disease state, or to identify autoantigens.
  • libraries that include an array of human polypeptides are prepared.
  • libraries that include polypeptides from infectious agents which are suspected of eliciting cross-reactive responses to autoantigens are prepared.
  • the present disclosure includes methods in which polypeptides of interest are included in a library (e.g., expressed in library cells or carried in or on particles or beads).
  • a library e.g., expressed in library cells or carried in or on particles or beads.
  • the polypeptides of interest are proteolytically processed within the antigen presenting cells, and peptide fragments of the polypeptides are presented on MEW molecules expressed in the antigen presenting cells.
  • the identity of the polypeptide that stimulates a human lymphocyte in an assay described herein can be determined from examination of the set of library cells that were provided to the antigen presenting cells that produced the stimulation.
  • This epitope, or the longer polypeptide from which it is derived both of which are referred to as an “antigen” herein
  • epitopes are identified by generating deletion mutants of the polypeptide of interest and testing these for the ability to stimulate lymphocytes. Deletions that lose the ability to stimulate lymphocytes, when processed and presented by antigen presenting cells, have lost the peptide epitope. In some embodiments, epitopes are identified by synthesizing peptides corresponding to portions of the polypeptide of interest and testing the peptides for the ability to stimulate lymphocytes (e.g., in antigen presentation assays in which antigen presenting cells are pulsed with the peptides).
  • MEW bound peptides involve lysis of the antigen presenting cells that include the antigenic peptide, affinity purification of the MEW molecules from cell lysates, and subsequent elution and analysis of peptides from the MHC (Falk, K. et al. Nature 351:290, 1991, and U.S. Pat. No. 5,989,565).
  • T cell receptors that have been expanded in response to the antigen.
  • Clonal T cell receptors are identified by DNA sequencing of the T cell receptor repertoire (Howie et al, 2015 Sci Trans Med 7:301). By identifying TCR specificity and function, TCRs can be transfected into other cell types and used in functional studies or for novel immunotherapies.
  • T cells responsive to a tumor antigen in a subject.
  • the isolated T cells can be expanded ex vivo and administered to a subject for cancer therapy or prophylaxis.
  • one or more immune responses of a subject are determined by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the subject; c) contacting the antigen presenting cells with lymphocytes from the subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells.
  • the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80
  • a test subject is (i) a cancer subject who has not received a cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy; or (iii) a subject who has not been diagnosed with a cancer.
  • a target subject is (i) a cancer subject who responds or has responded positively clinically (“responsive subject”) to a cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically (“non-responsive subject”) to a cancer therapy; (iii) a cancer subject who responds or has responded spontaneously to a cancer (“spontaneous target subject”); or (vi) a subject who has not been diagnosed with a cancer (“normal subject”).
  • lymphocyte stimulation, non-stimulation, inhibition and/or suppression, activation, and/or non-responsiveness is determined by assessing levels of one or more expressed or secreted cytokines or other immune mediators described herein.
  • levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control indicates lymphocyte stimulation.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • NG Neon Green
  • a level of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level indicates lymphocyte inhibition and/or suppression.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level indicates lymphocyte inhibition and/or suppression.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control indicates lymphocyte inhibition and/or suppression.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher or lower than a control level indicates lymphocyte activation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater or lower than the mean of a control level indicates lymphocyte activation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control indicates lymphocyte activation.
  • MADs median absolute deviations
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • NG Neon Green
  • a level of one or more expressed or secreted cytokines that is within about 20%, 15%, 10%, 5%, or less, of a control level indicates lymphocyte non-responsiveness or non-stimulation.
  • a level of one or more expressed or secreted cytokines that is less than 1 or 2 standard deviations higher or lower than the mean of a control level indicates lymphocyte non-responsiveness or non-stimulation.
  • a level of one or more expressed or secreted cytokines that is less than 1 or 2 median absolute deviations (MADs) higher or lower than a median response level to a control indicates lymphocyte non-responsiveness or non-stimulation.
  • MADs median absolute deviations
  • a subject response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more cytokines) and of the total number of tumor antigens (e.g., of all or a portion of different tumor antigens from the library) that stimulate, do not stimulate, inhibit and/or suppress, activate, or have no or minimal effect on production, expression or secretion of each member of the panel of cytokines.
  • cytokines e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more cytokines
  • tumor antigens e.g., of all or a portion of different tumor antigens from the library
  • the disclosure provides methods for obtaining a subject response profile from a test subject (a “subject response profile”).
  • the subject response profile of a test subject is obtained by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the test subject; c) contacting the antigen presenting cells with lymphocytes from the test subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells, to obtain the subject response profile.
  • the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000, or more tumor antigens.
  • the subject response profile can include a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, or to which lymphocytes are non-responsive.
  • the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • the subject response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate expression or secretion of one or more immune mediators, that inhibit and/or suppress expression or secretion of one or more immune mediators, and/or which do not, or minimally, affect expression or secretion of immune mediators.
  • the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • a subject response profile is compared to a corresponding response profile from a target subject, e.g. a cancer subject who responds and/or has responded clinically to a cancer therapy; a cancer subject who does not and/or has not responded clinically to a cancer therapy; a subject who has, or has had, spontaneous response to a cancer; or a subject who has not been diagnosed with a cancer (a “target response profile” of a target subject).
  • a target subject e.g. a cancer subject who responds and/or has responded clinically to a cancer therapy
  • a cancer subject who does not and/or has not responded clinically to a cancer therapy e.g. a cancer subject who does not and/or has not responded clinically to a cancer therapy
  • a subject who has, or has had, spontaneous response to a cancer e.g. a “target response profile” of a target subject.
  • the disclosure provides methods for obtaining a target response profile from a target subject.
  • the target response profile of a target subject is obtained by a) providing a library described herein that includes all or a portion of the same panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein) used to generate the subject response profile; b) contacting the library with antigen presenting cells from the target subject; c) contacting the antigen presenting cells with lymphocytes from the target subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to, one or more tumor antigens presented by one or more antigen presenting cells, to obtain the target response profile.
  • tumor antigens e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor anti
  • the target response profile includes a quantification, identification, and/or representation of the immune response of cells from the target subject to the same panel of tumor antigens included in the subject response profile.
  • the target response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, and/or to which lymphocytes are non-responsive.
  • the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • the target response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens identified by the methods of the disclosure, that stimulate expression and/or secretion of one or more immune mediators, that inhibit and/or suppress expression or secretion of one or more immune mediators, and/or which do not, or minimally, affect expression and/or secretion of immune mediators.
  • the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • a subject response profile is similar to the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that stimulate lymphocytes in the target response profile; if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that activate lymphocytes in the target response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymph
  • a subject response profile is dissimilar from the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile; if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that activate lymphocytes in the target response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are
  • a subject response profile is similar to the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that stimulate lymphocytes in the target response profile; if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%,
  • a subject response profile is dissimilar from the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%,or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%,
  • the target response profile can include a quantification, identification, and/or representation of one or more cytokines and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on cytokine production, expression and/or secretion.
  • tumor antigens e.g., of the same tumor antigens included in the subject response profile
  • the target response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all) of the cytokines included in the subject response profile) and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on production, expression and/or secretion of the panel of cytokines.
  • cytokines e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all) of the cytokines included in the subject response profile
  • tumor antigens e.g., of the same tumor antigens included in the subject response profile
  • a subject response profile is similar to the target response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that inhibit and/or suppress one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that inhibit and/or suppress expression and/or secretion of the same one or more cytokines included in the target response profile; and/or
  • a subject response profile is dissimilar from the target response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that inhibit and/or suppress expression and/or secretion of one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in the target response profile; and/or if the
  • the foregoing methods apply to subject response profiles and target response profiles obtained with libraries encoding polypeptides that are potential tumor antigens, as well as tumor antigens.
  • the disclosure provides methods of identifying a test subject, e.g., a cancer subject, for initiation, continuation, modification, and/or discontinuation or in some cases non-initiation of a cancer therapy (e.g., a cancer therapy described herein).
  • a cancer therapy e.g., a cancer therapy described herein.
  • such methods include comparing one or more immune responses of a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy) to one or more immune responses of a target subject, who may be: (i) a cancer subject who responds or has responded positively clinically (“responsive subject”) to the cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically (“non-responsive subject”) to the cancer therapy; (iii) a cancer subject who responds or has responded spontaneously to a cancer (“spontaneous subject”); and/or (vi) a subject
  • One or more immune responses of the test subject that are the same or similar to one or more immune responses of a responsive subject and/or dissimilar to one or more immune responses of a non-responsive subject indicates that the test subject should initiate and/or continue and/or modify (e.g., increase and/or combine with one or more other modalities) the cancer therapy.
  • One or more immune responses of the test subject that are dissimilar to one or more immune responses of a responsive subject and/or similar to (or same as) one or more immune responses of a non-responsive subject indicates that the cancer subject should not initiate and/or should discontinue and/or should modify (e.g., reduce and/or combine with one or more other modalities) the cancer therapy, and/or should initiate an alternative cancer therapy, or in some cases, no cancer therapy.
  • a subject response profile that is similar to a target response profile (of a responsive subject) indicates the test subject should initiate and/or continue and/or modify (e.g., increase and/or combine with one or more other modalities) the cancer therapy.
  • methods described herein include selecting a test subject for initiation and/or continuation and/or modification (e.g., increase and/or combine with one or more other modalities) of the cancer therapy if the subject response profile is similar to a target response profile (of a responsive subject).
  • methods described herein include initiating and/or continuing and/or modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a responsive subject). In some embodiments, methods described herein include administering the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a responsive subject). In some embodiments, methods described herein include modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a responsive subject).
  • a subject response profile that is dissimilar to a target response profile (of a responsive subject) indicates the test subject should not initiate and/or should modify (e.g., reduce and/or combine with one or more other modalities) and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy.
  • methods described herein include not selecting a test subject for initiation and/or selecting a test subject for modification (e.g., reduction and/or combination with one or more other modalities) and/or discontinuation of the cancer therapy and/or initiation of an alternative cancer therapy, if the subject response profile is dissimilar to a target response profile (of a responsive subject).
  • methods described herein include not initiating and/or modifying (e.g., reducing and/or combining with one or more other modalities) and/or discontinuing administration of the cancer therapy to a test subject and/or initiation of an alternative cancer therapy, if the subject response profile is dissimilar to a target response profile (of a responsive subject). In some embodiments, methods described herein include not administering the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a responsive subject).
  • methods described herein include modifying (e.g., reducing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a responsive subject). In some embodiments, methods described herein include administering an alternative cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a responsive subject).
  • a subject response profile is compared to a corresponding response profile from a cancer subject who has not responded and/or is not responding and/or responds negatively, clinically to the cancer therapy (a “target response profile” of a non-responsive subject).
  • the target response profile (of a non-responsive subject) is obtained by providing a library described herein that includes all or a portion of the same panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein or identified using a method described herein) used to generate the subject response profile; contacting the library with antigen presenting cells from the non-responsive subject; contacting the antigen presenting cells with lymphocytes from the non-responsive subject; and determining whether one or more lymphocytes are stimulated, inhibited and/or suppressed by, or non-responsive to, one or more tumor antigens presented by one or more antigen presenting cells.
  • the target response profile (of a non-responsive subject) includes a quantification, identification, and/or representation of
  • Methods for comparing a subject response profile to a target response profile, and parameters for determining similarity and dissimilarily of a subject response profile to a target response profile are provided in the disclosure.
  • the target response profile (of a non-responsive subject) includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, and/or that inhibit and/or suppress lymphocytes.
  • a subject response profile is similar to the target response profile (of a nonresponsive subject) if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a nonresponsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that do not stimulate lymphocytes in the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a nonresponsive subject).
  • a subject response profile is dissimilar from the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a nonresponsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a nonresponsive subject).
  • a subject response profile is similar to the target response profile (of a nonresponsive subject) if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a nonresponsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that do not stimulate lymphocytesin the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a non-responsive subject).
  • a subject response profile is dissimilar from the target response profile (of a non-responsive subject) if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a non-responsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a non-responsive subject).
  • the target response profile (of a non-responsive subject) can include a quantification, identification, and/or representation of one or more cytokines and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, and/or inhibit and/or suppress cytokine production, expression and/or secretion.
  • the target response profile (of a nonresponsive subject) can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all), of the cytokines included in the subject response profile) and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, and/or inhibit and/or suppress production, expression and/or secretion of the panel of cytokines.
  • cytokines e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all)
  • tumor antigens e.g., of the same tumor antigens included in the subject response profile
  • a subject response profile is similar to the target response profile (of a nonresponsive subject) if the total number of antigens that stimulate one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that stimulate the same one or more cytokines included in the target response profile (of a non-responsive subject); if the total number of antigens that do not stimulate one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile (of a nonresponsive subject); and/or if the total number of antigens that inhibit and/or suppress one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5,10, 15, 20, or 25 from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in the target response profile (of
  • a subject response profile is dissimilar from the target response profile (of a non-responsive subject) if the total number of antigens that stimulate one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, or more, from the total number of antigens that stimulate the same one or more cytokines included in the target response profile (of a non-responsive subject); if the total number of antigens that not stimulate one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, or more, from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile (of a non-responsive subject); and/or if the total number of antigens that inhibit and/or suppress one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in the target response profile (of a non-responsive subject)
  • a subject response profile that is dissimilar to a target response profile (of a non-responsive subject) indicates the test subject should initiate and/or continue and/or modify (e.g., increase and/or combine with one or more other modalities) the cancer therapy.
  • methods described herein include selecting a test subject for initiation and/or continuation and/or modification of (e.g., increasing and/or combining with one or more other modalities) the cancer therapy if the subject response profile is dissimilar to a target response profile (of a non-responsive subject).
  • methods described herein include initiating and/or continuing and/or modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a non-responsive subject).
  • methods described herein include administering the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a non-responsive subject).
  • methods described herein include modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a non-responsive subject).
  • a subject response profile that is similar to a target response profile (of a non-responsive subject) indicates the test subject should not initiate, and/or should modify (e.g., reduce and/or combine with one or more other modalities), and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy.
  • methods described herein include not selecting a test subject for initiation and/or selecting a test subject for modification (e.g., reduction and/or combination with one or more other modalities) and/or discontinuation of the cancer therapy and/or initiation of an alternative cancer therapy, if the subject response profile is similar to a target response profile (of a non-responsive subject).
  • methods described herein include not initiating and/or modifying (e.g., reducing and/or combining with one or more other modalities) and/or discontinuing administration of the cancer therapy to a test subject and/or initiating an alternative cancer therapy, if the subject response profile is similar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include not administering the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a non-responsive subject).
  • methods described herein include modifying (e.g., reducing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include administering an alternative cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a non-responsive subject).
  • a subject response profile described herein is compared to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) target response profiles of one or more responsive subjects and/or of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) non-responsive subjects.
  • a target response profile described herein e.g., of a responsive subject or non-responsive subject
  • one or more subject response profiles of the test subject are obtained (e.g., before, during, and/or after initiation, modification, and/or discontinuation of administration of the cancer therapy).
  • immune responses can be usefully defined in terms of their integrated, functional end-effects.
  • Dhabar et al. (2014) have proposed that immune responses can be categorized as being immunoprotective, immunopathological, and immunoregulatory/inhibitory. While these categories provide useful constructs with which to organize ideas, an overall in vivo immune response is likely to consist of several types of responses with varying amounts of dominance from each category.
  • Immunoprotective or beneficial responses are defined as responses that promote efficient wound healing, eliminate infections and cancer, and mediate vaccine-induced immunological memory. These responses are associated with cytokines and mediators such as IFN-gamma, IL-12, IL-2, Granzyme B, CD107, etc.
  • Immunopathological or deleterious responses are defined as those that are directed against self (autoimmune disease like multiple sclerosis, arthritis, lupus) or innocuous antigens (asthma, allergies) and responses involving chronic, non-resolving inflammation. These responses can also be associated with molecules that are implicated in immunoprotective responses, but also include immune mediators such as TNF-alpha, IL-10, IL-13, IL-17, IL-4, IgE, histamine, etc. Immunoregulatory responses are defined as those that involve immune cells and factors that regulate (mostly down-regulate) the function of other immune cells. Recent studies suggest that there is an arm of the immune system that functions to inhibit immune responses.
  • regulatory CD4 + CD25+FoxP3 + T cells, IL-10, and TGF-beta have been shown to have immunoregulatory/inhibitory functions.
  • the physiological function of these factors is to keep pro-inflammatory, allergic, and autoimmune responses in check, but they may also suppress anti-tumor immunity and be indicative of negative prognosis for cancer.
  • the expression of co-stimulatory molecules often decreases, and the expression of co-inhibitory ligands increases.
  • MEW molecules are often down-regulated on tumor cells, favoring their escape.
  • the tumor micro-environment including stromal cells, tumor associated immune cells, and other cell types, produce many inhibitory factors, such as, IL-10, TGF- ⁇ , and IDO.
  • Inhibitory immune cells including T regs, Tr1 cells, immature DCs (iDCs), pDCs, and MDSC can be found in the tumor microenvironment. (Y Li UT GSBS Thesis 2016). Examples of mediators and their immune effects are shown in Table 2.
  • X stimulates T cell phages, growth, induces IFN- neutron- gamma/TNF-alpha phils secretion from T cells, enhances CTLs IL-2 T cell proliferation, T cells, APCs X X X ? ? ? differentiation into effector and memory T cells and regulatory T cells TNF- Induces fevers, Macro- X X ? X ? X alpha apoptosis, phages, inflammation, APCs inhibits viral replication MIP-1 Chemotactic/pro- Macro- X X ? ? ?
  • X alpha inflammatory phages, DCs, effects activates T cells granulocytes, induces secretion of IL-1/IL6/TNF-alpha MIP-1
  • X beta inflammatory phages, DCs, effects activates T cells granulocytes, induces secretion of IL-1/IL6/TNF-alpha CXCL9 T cell APCs X X ? X ? X chemoattractant, induced by IFN-gamma CXCL10 Chemoattractant for APCs X X ? ? ?
  • T cells macrophages, NK and DCs, promotes T cell adhesion to endothelial cells
  • MCP-1 recruits monocytes, most cells X X ? X ? X memory T cells and DCS RANTES recruits T cells, T cells X X ? ? ? X eosinophils, basophils, induces proliferation/activation of NK cells, T cell activation marker CXCL11
  • T cells IL-17I Produced by Th17 T cells X X ? X ? X cells
  • T cells X X X X ? X IL-24 Controls survival and Monocytes- X X ? ? ? X proliferation macro- phages, Th2 cells IL-27 Induces APCs, T cells X X X X ? X differentiation of T cells, upregulates IL- 10, can be pro-or anti-inflammatory; promotes Th1/Tr1, inhibits Th2/Th17/ regulatory T cells IL-32 Pro-inflammatory, T cells, NK X X ? X ? X increases secretion cells of inflammatory cytokines and chemokines CSF Induces myeloid APCs X X X ? ? ?
  • X X X Th2 proliferation basophils plasma cell differentiation, IgE, upregulates MHC Class II expression, decreases IFN- gamma production IL-10 Down regulates Th1 Monocytes X ? X X X X cytokines/MHC Class Th2 cells, II expression/Co- regulatory T stimulatory molecule cells expression IL-5 Stimulates B cells, Ig Th2 cells, ? X ? X X X secretion, eosinophil mast cells activation IL-13 Similar to IL4, Th2 cells, NK ? X ?
  • X X X induces IgE cells, mast production, Th2 cells, cytokine eosinophils, basophils TGF-beta Inhibits T cell regulatory T ? ? X X X ? proliferation, cells activity, function; blocks effects of pro- inflammatory cytokines IL-1 beta Induces fevers, pro- Macro- X X ? X ? X inflammatory phages IL-6 Pro-inflammatory, T cells, ? X ? X X X drives osteoclast macro- formation, drives phages Th17 IL-8 recruits neutrophils Macro- ? X ? X ?
  • a tumor antigen stimulates one or more lymphocyte responses that are beneficial to the subject. In some embodiments, a tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to the subject.
  • immune responses include but are not limited to 1) cytotoxic CD8 + T cells which can effectively kill cancer cells and release the mediators perforin and/or granzymes to drive tumor cell death; and 2) CD4 + Th1 T cells which play an important role in host defense and can secrete IL-2, IFN-gamma and TNF-alpha. These are induced by IL-12, IL-2, and IFN gamma among other cytokines.
  • a tumor antigen stimulates one or more lymphocyte responses that are deleterious or non-beneficial to the subject.
  • a tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are beneficial to the subject.
  • immune responses that may lead to deleterious or non-beneficial anti-tumor responses include but are not limited to 1) T regulatory cells which are a population of T cells that can suppress an immune response and secrete immunosuppressive cytokines such as TGF-beta and IL-10 and express the molecules CD25 and FoxP3; and 2) Th2 cells which target responses against allergens but are not productive against cancer. These are induced by increased IL-4 and IL-10 and can secrete IL-4, IL-5, IL-6, IL-9 and IL-13.
  • methods and systems described herein can identify and select one or more tumor antigens to which one or more immune responses are stimulated in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy). In some embodiments, methods and systems described herein can identify and select one or more tumor antigens to which one or more immune responses are not stimulated in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy).
  • methods and systems described herein can identify and select one or more tumor antigens to which one or more immune responses are inhibited and/or suppressed in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy). In some embodiments, methods and systems described herein can identify and select one or more tumor antigens which elicit no or minimal immune responses in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy).
  • a composition comprising the one or more selected tumor antigens is administered to a cancer subject before, during, and/or after administration of a cancer therapy.
  • the disclosure provides methods for selecting tumor antigens identified by the methods herein based on comparison of a subject response profile to a target response profile.
  • the disclosure also provides methods for selecting (or de-selecting) tumor antigens identified by the methods herein, based on association with desirable or beneficial responses.
  • the disclosure also provides methods for selecting (or de-selecting) tumor antigens identified by the methods herein, based on association with undesirable, deleterious or non-beneficial responses.
  • the methods for selecting tumor antigens are combined. The methods may be combined in any order, e.g.
  • selection may be carried out by comparison of a subject response profile to a target response profile, followed by selection based on association with a desirable (or undesirable) response; or, selection may be carried out based on association with a desirable (or undesirable) response, followed by comparison of the subject response profile to a target response profile.
  • Methods for identifying tumor antigens and potential tumor antigens are provided herein.
  • Methods for generating or obtaining a subject response profile are provided herein.
  • Methods for generating or obtaining a target response profile e.g. a population-based or composite target response profile, are provided herein.
  • Methods for comparison of a subject response profile to a target response profile are provided herein.
  • Methods for determining whether a subject response profile is similar to a target response profile are provided herein.
  • a subject response profile and target response profile are generated or obtained using the same plurality of polypeptides of interest. In some embodiments, a subject response profile and target response profile are generated or obtained using the same plurality of tumor antigens.
  • the target response profile includes a quantification, identification, and/or representation of one or more tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, and/or to which lymphocytes are non-responsive.
  • one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the test subject (e.g., identified from the subject response profile), and the same one or more tumor antigens are identified as stimulating lymphocytes in the target subject (e.g., identified from the target response profile).
  • one or more tumor antigens are identified as stimulating lymphocytes in the test subject (e.g., identified from the subject response profile) and the same one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the target subject (e.g., identified from the target response profile).
  • one or more tumor antigens or potential tumor antigens are identified as eliciting minimal or no response from lymphocytes in the test subject (e.g., identified from the subject response profile), and the same one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing lymphocytes in the target subject (e.g., identified from the target response profile).
  • one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing, lymphocytes in the test subject (e.g., identified from the subject response profile), and the same one or more tumor antigens are identified as eliciting minimal or no response from lymphocytes in the target subject (e.g., identified from the target response profile).
  • Tumor antigens may be identified and/or selected on the basis of similarity or dissimilarity of a subject response profile to a target response profile. Tumor antigens may be identified and/or selected (or de-selected) based on association with desirable or beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on association with undesirable, deleterious or non-beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on a combination of the preceding methods, applied in any order.
  • a subject response profile is compared to a corresponding response profile from a cancer subject who responds and/or has responded clinically to a cancer therapy (a “target response profile” of a responsive subject described herein).
  • a subject response profile is compared to a target response profile from a target subject who has not been diagnosed with cancer.
  • a subject response profile is compared to a target response profile from a target subject who has (or had) a beneficial response to cancer.
  • the subject has (or had) a positive clinical response to a cancer therapy or combination of therapies.
  • the subject had a spontaneous response to a cancer.
  • the subject is in partial or complete remission from cancer.
  • the subject has cleared a cancer. In some embodiments, the subject has not had a relapse, recurrence or metastasis of a cancer. In some embodiments, the subject has a positive cancer prognosis. In some embodiments, the subject has not experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • one or more tumor antigens of the subject response profile which elicit responses that are different from, or dissimilar to, responses elicited by the same tumor antigens of the target response profile are selected. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with desirable or beneficial immune responses. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with undesirable, deleterious, or non-beneficial immune responses.
  • tumor antigens or immunogenic fragments thereof stimulate lymphocyte responses that are beneficial to the subject, (ii) stimulate expression of cytokines that are beneficial to the subject, (iii) inhibit and/or suppress lymphocyte responses that are deleterious or non-beneficial to the subject, or (iv) inhibit and/or suppress expression of cytokines that are deleterious or non-beneficial to the subject, are termed “beneficial responses”.
  • a selected tumor antigen stimulates one or more lymphocyte responses that are beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to the subject.
  • a selected tumor antigen increases expression and/or secretion of cytokines that are beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses expression of cytokines that are deleterious or non-beneficial to the subject.
  • administration of one or more selected tumor antigens to the subject elicits an immune response of the subject. In some embodiments, administration of one or more selected tumor antigens to the subject elicits a beneficial immune response of the subject. In some embodiments, administration of one or more selected tumor antigens to the subject elicits a beneficial response of the subject. In some embodiments, administration of one or more selected tumor antigens to the subject improves clinical response of the subject to a cancer therapy.
  • a subject response profile is compared to a corresponding response profile from a cancer subject who does not respond and/or has not responded clinically to a cancer therapy (a “target response profile” of a non-responsive subject described herein).
  • a subject response profile is compared to a target response profile from a target subject who has (or had) a deleterious or non-beneficial response to cancer.
  • the subject has (or had) a negative clinical response to a cancer therapy or combination of therapies.
  • the subject has not cleared a cancer.
  • the subject has had a relapse, recurrence or metastasis of a cancer.
  • the subject has a negative cancer prognosis.
  • the subject has experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • tumor antigens or immunogenic fragments thereof stimulate lymphocyte responses that are deleterious or not beneficial to the subject, (ii) stimulate expression of cytokines that are deleterious or not beneficial to the subject, (iii) inhibit and/or suppress lymphocyte responses that are beneficial to the subject, or (iv) inhibit and/or suppress expression of cytokines that are beneficial to the subject, are termed “deleterious or non-beneficial responses”.
  • one or more tumor antigens of the subject response profile which elicit responses that are the same as, or similar to, responses elicited by the same tumor antigens of the target response profile are selected. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with desirable or beneficial immune responses. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with undesirable, deleterious, or non-beneficial immune responses.
  • a selected tumor antigen stimulates one or more lymphocyte responses that are deleterious or non-beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are beneficial to the subject.
  • a selected tumor antigen increases expression and/or secretion of cytokines that are deleterious or non-beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses expression of cytokines that are beneficial to the subject.
  • the one or more tumor antigens are de-selected by the methods herein.
  • the one or more selected tumor antigens are excluded from administration to a subject.
  • T cells that have been educated in the context of the tumor microenvironment sometimes are sub-optimally activated, have low avidity, and ultimately fail to recognize the tumor cells that express antigen.
  • tumors are complex and comprise numerous cell types with varying degrees of expression of mutated genes, making it difficult to generate polyclonal T cell responses that are adequate to control tumor growth.
  • researchers in the field have proposed that it is important in cancer subjects to identify the mutations that are “potential tumor antigens” in addition to those that are confirmed in the cancer subject to be recognized by their T cells.
  • the present disclosure provides methods to a) identify polypeptides that are potential tumor antigens in antigen presentation assays of the disclosure, and b) select polypeptides on the basis of their antigenic potential.
  • the methods are performed without making predictions about what could be a target of T cell responses or presented by MHC, and without the need for deconvolution.
  • the methods can be expanded to explore antigenic potential in healthy subjects who share the same MHC alleles as a subject, to identify those potential tumor antigens that would be most suitable to include in an immunogenic composition or vaccine formulation.
  • the methods ensure that the potential tumor antigen is processed and presented in the context of subject MHC molecules, and that T cells can respond to the potential tumor antigen if they are exposed to the potential tumor antigen under the right conditions (e.g., in the context of a vaccine with a strong danger signal from an adjuvant or delivery system).
  • the preceding methods for selection of tumor antigens may be applied to selection of potential tumor antigens, that is, polypeptides encoding one or more mutations present or expressed in a cancer or tumor cell of a subject.
  • compositions that include a tumor antigen or tumor antigens identified or selected by methods described herein, nucleic acids encoding the tumor antigens, and methods of using the compositions.
  • a composition includes tumor antigens that are peptides 8-40 amino acids, 8-60 amino acids, 8-100. 8-150,or 8-200 amino acids in length (e.g., MHC binding peptides, e.g., peptides 23-29, 24-28, 25-27, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-15, 8-12 amino acids in length).
  • a composition includes one or more tumor antigens that are about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the full-length polypeptides.
  • a composition includes one or more tumor antigens that are truncated by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids, relative to the full-length polypeptides.
  • the compositions can include tumor antigens that are, or that comprise, MHC class I-binding peptides, MHC class II-binding peptides, or both MHC class I and MHC class II-binding peptides.
  • Compositions can include a single tumor antigen, or multiple tumor antigens.
  • a composition includes a set of two, three, four, five, six, seven, eight, nine, ten, or more tumor antigens.
  • a composition includes ten, fifteen, twenty, twenty-five, thirty, or more tumor antigens.
  • the tumor antigens or peptides are provided as one or more fusion proteins.
  • a composition comprises nucleic acids encoding the tumor antigens or peptides.
  • the nucleic acids encoding the tumor antigens or peptides are provided as one or more fusion constructs.
  • compositions comprising any combination of two or three TAAs: HPSE1 (SEQ ID NO: 6), HPSE2 (SEQ ID NO: 7), and/or SMAD4 (SEQ ID NO: 8).
  • HPSE encodes Heparinase, an endoglycosidase that cleaves heparan sulfate proteoglycans (HSPGs) into heparan sulfate side chains and core proteoglycans.
  • HPSE participates in extracellular matrix (ECM) degradation and remodeling.
  • ECM extracellular matrix
  • HPSE isoform 1 HPSE1
  • HPSE2 HPSE2
  • HPSE2 has no enzymatic activity, but may regulate HPSE1 activity.
  • the active protein form of HPSE1 is a heterodimer of 8 and 50 kDa subunits which are non-covalently linked.
  • the TIM barrel fold domain contains the active site, and the C-terminal domain of the protein is involved in nonenzymatic signaling and secretory functions.
  • Potential T-cell epitopes within HPSE have been described (Tang. In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies. Cancer Sci 105 (2014) 9-17).
  • the protein sequences of HPSE1 and HPSE2 may be found by searching in the publicly available database, UniProt (on the World Wide Web, at http://www.uniprot.org/uniprot/Q9Y251) and http://www.uniprot.org/uniprot/Q8WWQ2 respectively).
  • HPSE1 and HPSE2 may be found by searching in the publicly available database, Entrez (on the World Wide Web https://www.ncbi.nlm.nih.gov/gene/10855 and https://www.ncbi.nlm.nih.gov/gene/60495 respectively).
  • SMAD4 encodes Mothers against decapentaplegic homolog 4, a signal transduction protein and tumor suppressor gene, which is a central mediator of downstream transcriptional output in TGFb signaling pathways.
  • SMAD4 is a 552 amino acid, 60.4 KDa protein.
  • SMAD4 exists as a monomer in the absence of TGF-beta activation, and a heterodimer on TGF-beta activation.
  • SMAD4 is composed of two molecules of a C-terminally phosphorylated R-SMAD molecule, SMAD2 or SMAD3, and one molecule of SMAD4 to form the transcriptional active SMAD2/SMAD3-SMAD4 complex.
  • SMAD4 regulates transcription of a number of target genes through binding to DNA, recognizing an 8-bp palindromic sequence (GTCTAGAC) called the Smad-binding element (SBE).
  • GTCTAGAC 8-bp palindromic sequence
  • SBE Smad-binding element
  • the protein acts as a tumor suppressor and inhibits epithelial cell proliferation.
  • the protein and DNA sequences of SMAD4 may be found by searching in the publicly available databases, UniProt and Entrez (on the World Wide Web, at http://www.uniprot.org/uniprot/Q13485 and https://www.ncbi.nlm.nih.gov/gene/4089 respectively).
  • the disclosure also provides nucleic acids encoding the tumor antigens.
  • the nucleic acids can be used to produce expression vectors, e.g., for recombinant production of the tumor antigens, or for nucleic acid-based administration in vivo (e.g., DNA vaccination).
  • tumor antigens are used in diagnostic assays.
  • compositions including the tumor antigens can be provided in kits, e.g., for detecting antibody reactivity, or cellular reactivity, in a sample from an individual.
  • tumor antigen compositions are used to induce an immune response in a subject.
  • the subject is a human.
  • the subject is a non-human animal.
  • the tumor antigen compositions can be used to raise antibodies (e.g., in a non-human animal, such as a mouse, rat, hamster, or goat), e.g., for use in diagnostic assays, and for therapeutic applications.
  • a tumor antigen discovered by a method described herein may be a potent T cell and/or B cell antigen.
  • Preparations of antibodies may be produced by immunizing a subject with the tumor antigen and isolating antiserum from the subject.
  • the tumor antigen compositions are used to raise monoclonal antibodies, e.g., human monoclonal antibodies.
  • a tumor antigen composition is used to induce an immune response in a human subject to provide a therapeutic response.
  • a tumor antigen composition is used to induce an immune response in a human subject that redirects an undesirable immune response.
  • a tumor antigen composition elicits an immune response that causes the subject to have a positive clinical response described herein, e.g., as compared to a subject who has not been administered the tumor antigen composition.
  • a tumor antigen composition elicits an immune response that causes the subject to have an improved clinical response, e.g., as compared to a subject who has not been administered the tumor antigen composition.
  • a tumor antigen composition is used to induce an immune response in a human subject for palliative effect. The response can be complete or partial therapy.
  • a tumor antigen composition is used to induce an immune response in a human subject to provide a prophylactic response.
  • the response can be complete or partial protection.
  • immunogenicity of a tumor antigen is evaluated in vivo.
  • humoral responses to a tumor antigen are evaluated (e.g., by detecting antibody titers to the administered tumor antigen).
  • cellular immune responses to a tumor antigen are evaluated, e.g., by detecting the frequency of antigen-specific cells in a sample from the subject (e.g., by staining T cells from the subject with WIC/peptide tetramers containing the antigenic peptide, to detect antigen-specific T cells, or by detecting antigen-specific cells using an antigen presentation assay such as an assay described herein).
  • the ability of a tumor antigen or antigens to elicit protective or therapeutic immunity is evaluated in an animal model. In some embodiments, the ability of a tumor antigen or antigens to stimulate or to suppress and/or inhibit immunity is evaluated in an animal model.
  • the composition includes a pharmaceutically acceptable carrier or excipient.
  • An immunogenic composition may also include an adjuvant for enhancing the immunogenicity of the formulation, (e.g., oil in water, incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, saponin adjuvants, toll-like receptor agonists, or muramyl dipeptides). Other adjuvants are known in the art.
  • an immunogenic composition includes a tumor antigen linked to a carrier protein.
  • carrier proteins include, e.g., toxins and toxoids (chemical or genetic), which may or may not be mutant, such as anthrax toxin, PA and DNI (PharmAthene, Inc.), diphtheria toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.) or CRM 197, tetanus toxin, tetanus toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.), tetanus toxin fragment Z, exotoxin A or mutants of exotoxin A of Pseudomonas aeruginosa , bacterial flagellin, pneumolysin, an outer membrane protein of Neisseria meningitidis (strain available from the ATCC (American Type Culture Collection, Manassas, Va.)), Ps
  • coli heat labile enterotoxin shiga-like toxin
  • human LTB protein a protein extract from whole bacterial cells, and any other protein that can be cross-linked by a linker.
  • Other useful carrier proteins include high density lipoprotein (HDL), bovine serum albumin (BSA), P40, and chicken riboflavin. Many carrier proteins are commercially available (e.g., from Sigma Aldrich.).
  • an immunogenic composition including a tumor antigen identified by a method described herein is used in conjunction with an available vaccine.
  • an antigen identified as described herein can be used as a supplemental component of a vaccine formulation, or as a boosting antigen in a vaccination protocol.
  • an immunogenic composition is in a volume of about 0.5 mL for subcutaneous injection, 0.1 mL for intradermal injection, or 0.002-0.02 mL for percutaneous administration.
  • a 0.5 ml dose of the composition may contain approximately 2-500 ug of the tumor antigen.
  • an immunogenic composition is administered parenterally (for instance, by subcutaneous, intramuscular, intravenous, or intradermal injection).
  • delivery by a means that physically penetrates the dermal layer is used (e.g., a needle, airgun, or abrasion).
  • an immunogenic composition is administered to a subject, e.g., by intramuscular injection, intradermal injection, or transcutaneous immunization with appropriate immune adjuvants.
  • Compositions can be administered, one or more times, often including a second administration designed to boost an immune response in a subject.
  • the frequency and quantity of dosage of the composition can vary depending on the specific activity of the composition and clinical response of the subject, and can be determined by routine experimentation.
  • the formulations of immunogenic compositions can be provided in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier immediately prior to use.
  • a tumor antigen suitable for use in any method or composition of the disclosure may be produced by any available means, such as recombinantly or synthetically (see, e.g., Jaradat Amino Acids 50:39-68 (2016); Behrendt et al., J. Pept. Sci. 22:4-27 (2016)).
  • a tumor antigen may be recombinantly produced by utilizing a host cell system engineered to express a tumor antigen-encoding nucleic acid.
  • a tumor antigen may be produced by activating endogenous genes.
  • a tumor antigen may be partially or fully prepared by chemical synthesis.
  • any expression system can be used.
  • known expression systems include, for example, E. coli , egg, baculovirus, plant, yeast, or mammalian cells.
  • recombinant tumor antigen suitable for the present invention are produced in mammalian cells.
  • mammalian cells that may be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J.
  • human fibrosarcoma cell line e.g., HT1080
  • baby hamster kidney cells BHK21, ATCC CCL 10
  • Chinese hamster ovary cells+/ ⁇ DHFR CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980
  • mouse sertoli cells TM4, Mather, Biol.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • the present invention provides recombinant tumor antigen produced from human cells. In some embodiments, the present invention provides recombinant tumor antigen produced from CHO cells or HT1080 cells.
  • cells that are engineered to express a recombinant tumor antigen may comprise a transgene that encodes a recombinant tumor antigen described herein.
  • the nucleic acids encoding recombinant tumor antigen may contain regulatory sequences, gene control sequences, promoters, non-coding sequences and/or other appropriate sequences for expressing the recombinant tumor antigen.
  • the coding region is operably linked with one or more of these nucleic acid components.
  • the coding region of a transgene may include one or more silent mutations to optimize codon usage for a particular cell type.
  • the codons of a tumor antigen transgene may be optimized for expression in a vertebrate cell.
  • the codons of a tumor antigen transgene may be optimized for expression in a mammalian cell.
  • the codons of a tumor antigen transgene may be optimized for expression in a human cell.
  • the disclosure provides methods of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: a) providing, preparing, or obtaining a plurality of antigenic compositions comprising a plurality of antigens, each composition comprising a different antigen; b) providing, preparing, or obtaining a target response profile, wherein the target response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; c) providing, preparing, or obtaining a subject response profile, wherein the subject response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; d) comparing the target response profile to the subject response profile; e) selecting one or more antigens based on the comparison; and f) formulating at least a portion of one or more antigenic compositions
  • antigenic compositions are provided, prepared, or obtained.
  • a plurality of antigens can be produced using a method described herein, e.g., recombinantly or synthetically.
  • the antigens can be provided in a suitable composition, such as a solution or lyophilized composition.
  • the antigens are synthetically produced.
  • a synthetically produced antigen remains attached to a solid support.
  • formulating an antigen includes aliquoting a portion of the antigenic composition, reconstituting at least a portion of a lyophilized antigenic composition, and/or releasing a synthetically produced antigen from a solid support.
  • Antigenic compositions may be prepared or obtained and stored in a variety of forms, such as in a suspension, in solution, or lyophilized. Antigenic compositions may be stored at a temperature ranging from less than ⁇ 80° C. to about room temperature, for example at about ⁇ 80° C., about ⁇ 20° C., about ⁇ 15° C., about ⁇ 10° C., about 4° C. or at about room temperature. In some embodiments, antigenic compositions may include a carrier, excipient, stabilizer, preservative and/or adjuvant.
  • a plurality of antigens can be derived from a target response profile wherein the target response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated with (e.g., determined, measured, observed) with the plurality of antigens.
  • a plurality of antigens can be derived from a subject response profile wherein the subject response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated with (e.g., determined, measured, observed) with the plurality of antigens.
  • a target response profile and subject response profile are compared and one or more antigens are selected based on the comparision.
  • one or more antigens are selected that increase expression or secretion of immune mediators associated with a beneficial response to cancer, and/or one or more antigens that inhibit and/or suppress expression or secretion of immune mediators associated with deleterious or not beneficial responses to cancer.
  • the selected antigens, or a portion of the selected antigens may be formulated as a pharmaceutical composition.
  • a tumor is or comprises a hematologic malignancy, including but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Langerhans cell histiocytosis, multiple myeloma, or myeloproliferative neoplasms.
  • a tumor is or comprises a solid tumor, including but not limited to breast carcinoma, a squamous cell carcinoma, a colon cancer, a head and neck cancer, ovarian cancer, a lung cancer, mesothelioma, a genitourinary cancer, a rectal cancer, a gastric cancer, or an esophageal cancer.
  • a tumor is or comprises an advanced tumor, and/or a refractory tumor.
  • a tumor is characterized as advanced when certain pathologies are observed in a tumor (e.g., in a tissue sample, such as a biopsy sample, obtained from a tumor) and/or when cancer patients with such tumors are typically considered not to be candidates for conventional chemotherapy.
  • pathologies characterizing tumors as advanced can include tumor size, altered expression of genetic markers, invasion of adjacent organs and/or lymph nodes by tumor cells.
  • a tumor is characterized as refractory when patients having such a tumor are resistant to one or more known therapeutic modalities (e.g., one or more conventional chemotherapy regimens) and/or when a particular patient has demonstrated resistance (e.g., lack of responsiveness) to one or more such known therapeutic modalities.
  • one or more known therapeutic modalities e.g., one or more conventional chemotherapy regimens
  • resistance e.g., lack of responsiveness
  • the present disclosure provides methods and systems related to cancer therapy.
  • the present disclosure is not limited to any specific cancer therapy, and any known or developed cancer therapy is encompassed by the present disclosure.
  • Known cancer therapies include, e.g., administration of chemotherapeutic agents, radiation therapy, surgical excision, chemotherapy following surgical excision of tumor, adjuvant therapy, localized hypothermia or hyperthermia, anti-tumor antibodies, and anti-angiogenic agents.
  • cancer and/or adjuvant therapy includes a TLR agonist (e.g., CpG, Poly I:C, etc., see, e.g., Wittig et al., Crit. Rev. Oncol. Hematol.
  • the cancer therapy is or comprises oncolytic virus therapy, e.g., talimogene leherparepvec. (see, e.g., Fukuhara et al., Cancer Sci. 107:1373-1379 (2016)).
  • the cancer therapy is or comprises bi-specific antibody therapy (e.g., Choi et al., 2011 Expert Opin Biol Ther ; Huehls et al., 2015 , Immunol and Cell Biol).
  • the cancer therapy is or comprises cellular therapy such as chimeric antigen receptor T (CAR-T) cells, TCR-transduced T cells, dendritic cells, tumor infiltrating lymphocytes (TIL), or natural killer (NK) cells (e.g., as reviewed in Sharpe and Mount, 2015 , Dis Model Mech 8:337-50).
  • CAR-T chimeric antigen receptor T
  • TCR-transduced T cells TCR-transduced T cells
  • dendritic cells dendritic cells
  • TIL tumor infiltrating lymphocytes
  • NK natural killer cells
  • Anti-tumor antibody therapies i.e., therapeutic regimens that involve administration of one or more anti-tumor antibody agents
  • Antibody agents have been designed or selected to bind to tumor antigens, particularly those expressed on tumor cell surfaces.
  • useful anti-tumor antibody agents see, for example, Adler et al., Hematol. Oncol. Clin. North Am. 26:447-81 (2012); Li et al., Drug Discov. Ther. 7:178-84 (2013); Scott et al., Cancer Immun. 12:14 (2012); and Sliwkowski et al., Science 341:1192-1198 (2013)).
  • Table 8 presents a non-comprehensive list of certain human antigens targeted by known, available antibody agents, and notes c
  • a cancer therapy is or comprises immune checkpoint blockade therapy (see, e.g., Martin-Liberal et al., Cancer Treat. Rev. 54:74-86 (2017); Menon et al., Cancers (Basel) 8:106 (2016)), or immune suppression blockade therapy.
  • Certain cancer cells thrive by taking advantage of immune checkpoint pathways as a major mechanism of immune resistance, particularly with respect to T cells that are specific for tumor antigens. For example, certain cancer cells may overexpress one or more immune checkpoint proteins responsible for inhibiting a cytotoxic T cell response.
  • immune checkpoint blockade therapy may be administered to overcome the inhibitory signals and permit and/or augment an immune attack against cancer cells.
  • Immune checkpoint blockade therapy may facilitate immune cell responses against cancer cells by decreasing, inhibiting, or abrogating signaling by negative immune response regulators (e.g., CTLA-4).
  • a cancer therapy or may stimulate or enhance signaling of positive regulators of immune response (e.g., CD28).
  • immune checkpoint blockade and immune suppression blockade therapy include agents targeting one or more of A2AR, B7-H4, BTLA, CTLA-4, CD28, CD40, CD137, GITR, IDO, KIR, LAG-3, PD-1, PD-L1, OX40, TIM-3, and VISTA.
  • immune checkpoint blockade agents include the following monoclonal antibodies: ipilimumab (targets CTLA-4); tremelimumab (targets CTLA-4); atezolizumab (targets PD-L1); pembrolizumab (targets PD-1); nivolumab (targets PD-1); avelumab; durvalumab; and cemiplimab.
  • immune suppression blockade agents include: Vista (B7-H5, v-domain Ig suppressor of T cell activation) inhibitors; Lag-3 (lymphocyte-activation gene 3, CD223) inhibitors; IDO (indolemamine-pyrrole-2,3,-dioxygenase-1,2) inhibitors; KIR receptor family (killer cell immunoglobulin-like receptor) inhibitors; CD47 inhibitors; and Tigit (T cell immunoreceptor with Ig and ITIM domain) inhibitors.
  • a cancer therapy is or comprises immune activation therapy.
  • immune activators include: CD40 agonists; GITR (glucocorticoid-induced TNF-R-related protein, CD357) agonists; OX40 (CD134) agonists; 4-1BB (CD137) agonists; ICOS (inducible T cell stimulator); CD278 agonists; IL-2 (interleukin 2) agonists; and interferon agonists.
  • cancer therapy is or comprises a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, or a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, and other cancer therapies.
  • the present disclosure provides methods and systems related to subjects who do not respond and/or have not responded; or respond and/or have responded (e.g., clinically responsive, e.g., clinically positively responsive or clinically negatively responsive) to a cancer therapy.
  • subjects respond and/or have responded positively clinically to a cancer therapy.
  • subjects respond and/or have responded negatively clinically to a cancer therapy.
  • subjects do not respond and/or have not responded (e.g., clinically non-responsive) to a cancer therapy.
  • Whether a subject responds positively, responds negatively, and/or fails to respond to a cancer therapy can be measured and/or characterized according to particular criteria.
  • criteria can include clinical criteria and/or objective criteria.
  • techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MM, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology.
  • a positive response, a negative response, and/or no response, of a tumor to a therapy can be assessed by ones skilled in the art using a variety of established techniques for assessing such response, including, for example, for determining one or more of tumor burden, tumor size, tumor stage, etc. Methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:e143-52.
  • a responsive subject exhibits a decrease in tumor burden, tumor size, and/or tumor stage upon administration of a cancer therapy. In some embodiments, a non-responsive subject does not exhibit a decrease in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy. In some embodiments, a non-responsive subject exhibits an increase in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy.
  • a cancer subject is identified and/or selected for administration of a cancer therapy as described herein.
  • the cancer therapy is administered to the subject.
  • the subject upon administration of the cancer therapy, the subject exhibits a positive clinical response to the cancer therapy, e.g., exhibits an improvement based on one or more clinical and/or objective criteria (e.g., exhibits a decrease in tumor burden, tumor size, and/or tumor stage).
  • the clinical response is more positive than a clinical response to the cancer therapy administered to a cancer subject who is identified (using a method described herein) as a cancer subject who should not initiate, and/or should modify (e.g., reduce and/or combine with one or more other modalities), and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy.
  • Methods described herein can include preparing and/or providing a report, such as in electronic, web-based, or paper form.
  • the report can include one or more outputs from a method described herein, e.g., a subject response profile described herein.
  • a report is generated, such as in paper or electronic form, which identifies the presence or absence of one or more tumor antigens (e.g., one or more stimulatory and/or inhibitory and/or suppressive tumor antigens, or tumor antigens to which lymphocytes are not responsive, described herein) for a cancer patient, and optionally, a recommended course of cancer therapy.
  • the report includes an identifier for the cancer patient.
  • the report is in web-based form.
  • a report includes information on prognosis, resistance, or potential or suggested therapeutic options.
  • the report can include information on the likely effectiveness of a therapeutic option, the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to a cancer patient, e.g., identified in the report.
  • the report can include information, or a recommendation, on the administration of a cancer therapy, e.g., the administration of a pre-selected dosage or in a pre-selected treatment regimen, e.g., in combination with one or more alternative cancer therapies, to the patient.
  • the report can be delivered, e.g., to an entity described herein, within 7, 14, 21, 30, or 45 days from performing a method described herein.
  • the report is a personalized cancer treatment report.
  • a report is generated to memorialize each time a cancer subject is tested using a method described herein.
  • the cancer subject can be reevaluated at intervals, such as every month, every two months, every six months or every year, or more or less frequently, to monitor the subject for responsiveness to a cancer therapy and/or for an improvement in one or more cancer symptoms, e.g., described herein.
  • the report can record at least the treatment history of the cancer subject.
  • the method further includes providing a report to another party.
  • the other party can be, for example, the cancer subject, a caregiver, a physician, an oncologist, a hospital, clinic, third-party payor, insurance company or a government office.
  • Example 1 Immune Responses to the ATLAS Melanoma Tumor Associated Antigen (TAA) Library—Single Patient Responses
  • a peripheral blood sample was collected from a consented melanoma patient who had previously undergone therapy with a checkpoint inhibitor (pembrolizumab) and responded to therapy.
  • Peripheral blood mononuclear cells PBMC
  • CD4 + and CD8 + T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation.
  • Monocytes were differentiated into dendritic cells (MDDC).
  • FIG. 1 shows representative results for a single melanoma patient.
  • Clones that induced mean IFN ⁇ responses that were statistically different from background (Wilcoxon Rank Sum, p ⁇ 0.05) and exceeded 3 standard deviations (SD) of the mean of the negative control GFP clones (N 10) were considered antigens (indicated by horizontal dotted line).
  • CEF positive control peptide pool.
  • Dozens of subjects were recruited into the study and cohorted based upon their clinical outcome after checkpoint inhibitor therapy. Subjects who had stable disease or tumor regression were considered protected; those who had worsening disease (tumor growth) were considered not protected. Clinical determinations were made by tumor imaging scans.
  • PBMC Peripheral blood mononuclear cells
  • CD4 + and CD8 + T cells were sorted and non-specifically expanded using anti-CD3 and anti-CD28-coated microbeads, and CD14 + monocytes were differentiated into dendritic cells (MDDC).
  • Library clones comprising known TAAs (labelled as Un001-023, as shown above in Table 3) were screened in duplicate using 5,000 MDDC and 80,000 T cells, at an E. coli :MDDC ratio of 100:1; ten replicates of E. coli expressing GFP were included as negative controls.
  • Assay supernatants were harvested at 24 hours and stored at ⁇ 80° C.
  • Supernatant cytokines were analyzed using Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
  • Clones that induced mean cytokine responses that were statistically different from background (Wilcoxon Rank Sum, p ⁇ 0.05) and exceeded 3 standard deviations (SD) of the mean responses to the negative control GFP clones (N 10) were considered antigens.
  • FIG. 2 shows cohort data for the CD4 + T cell subset. Subjects were cohorted into “Responder” (gray bars) or “Non-Responder” (black bars) groups based on clinical evaluation of disease. Using a cutoff of 3 SD above the mean of the negative control response per patient for each cytokine evaluated, the number of TAAs to which each subject responded with their CD4 + T cell subset is represented. In contrast to the Responder cohort, the Non-Responder group had minimal discernable CD4 + T cell responses, by the majority of cytokines evaluated, to any of the TAAs included in the library. Data are shown as the mean number ( ⁇ SE) of TAAs to which each cohort responded with each cytokine measured.
  • ⁇ SE mean number
  • FIG. 3 shows cohort data for the CD8 + T cell subset. Subjects were cohorted into “Responder” (gray bars) or “Non-Responder” (black bars) groups based on clinical evaluation of disease. Using a cutoff of 3 SD above the mean of the negative control response per patient for each cytokine evaluated, the number of TAAs to which each subject responded with their CD8 + T cell subset is represented. CD8 + T cells secreting IFN ⁇ were undetectable in Non-Responders, but Responders had responses to a mean of ⁇ two TAAs. Data are shown as the mean number ( ⁇ SE) of TAAs to which each cohort responded with each cytokine measured.
  • ⁇ SE mean number
  • ATLAS Generic Acids : Genocea Biosciences was applied to screen the entire complement of mutations identified in the tumor of a consented NSCLC patient who was successfully treated with pembrolizumab ( ⁇ PD-1 antibody (Ab), every other week starting on day 0).
  • An ATLAS library was built that expressed 201 of 202 mutations unique to this patient. Each clone contained 113 amino acids with the mutation positioned near the center of the construct and sequence-verified. Each clone was recombinantly expressed in E. coli .
  • Protein expression was verified using a surrogate T cell assay (the B3Z hybridoma) which recognizes the C57BL/6 mouse T cell epitope SIINFEKL (SEQ ID NO: 452), which is inserted at the C-terminus of each open reading frame, upstream of the stop codon. Proteins that induced B3Z responses that exceeded 5% of the positive control (the minimal SIINFEKL (SEQ ID NO: 452) epitope pulsed onto antigen presenting cells) were considered expressed.
  • the B3Z hybridoma which recognizes the C57BL/6 mouse T cell epitope SIINFEKL (SEQ ID NO: 452), which is inserted at the C-terminus of each open reading frame, upstream of the stop codon.
  • Proteins that induced B3Z responses that exceeded 5% of the positive control the minimal SIINFEKL (SEQ ID NO: 452) epitope pulsed onto antigen presenting cells
  • Peripheral blood samples were collected from the NSCLC patient before and after checkpoint blockade therapy.
  • Peripheral blood mononuclear cells PBMC
  • CD4 + and CD8 + T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation.
  • Monocytes were differentiated into dendritic cells (MDDC).
  • CD4 + and CD8 + T cells from Day 0 and Day 42 (after 3 rd injection) of treatment were screened, respectively, against 195 and 201 of the 201 library clones, as well as against 20 negative control clones expressing Neon Green (NG).
  • Library clones were screened in duplicate using 2,000 MDDC and 80,000 T cells, at an E. coli :MDDC ratio of 250:1. After 24h incubation, assay supernatants were harvested and stored at ⁇ 80° C. Supernatant cytokines were analyzed using a Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
  • FIG. 4 shows good alignment between duplicate measurements of the cytokines IFN ⁇ and TNF ⁇ for CD8 + T cell response, with over 74% of replicates falling within 1.5-fold of one another.
  • FIG. 5 shows results for IFN ⁇ and TNF ⁇ for CD8 + T cells pre- and post-treatment (left and right panels respectively).
  • NSCLC patient 5% of mutations screened (9 of 201) were identified as neoantigens recognized by his/her peripheral blood CD8 + T cells taken pre- and post-treatment. Only 1% of the identified neoantigens were found both pre- and post-treatment. Points above the top dotted line indicate neoantigens that stimulate CD8 + T cell responses. Points below the lower dotted line indicate neoantigens that suppress and/or inhibit CD8 + T cell responses.
  • FIG. 6 shows results for IFN ⁇ and TNF ⁇ for CD4 + T cells pre- and post-treatment (left and right panels respectively).
  • 10% of mutations screened (20 of 195) were identified as neoantigens recognized by his/her peripheral blood CD4 + T cells taken pre-treatment, increasing to 17% of mutations screened (33 of 195) post-treatment.
  • Five percent of the identified neoantigens were found both pre- and post-treatment. Points above the top dotted line indicate neoantigens that stimulate CD4 + T cell responses. Points below the lower dotted line indicate neoantigens that suppress and/or inhibit CD4 + T cell responses.
  • FIG. 7 shows the limited overlap between CD8 + -specific T cell neoantigens identified by ATLAS and epitope prediction algorithms.
  • MEW class I epitopes were predicted for all screened neoantigens from the NSCLC patient using three commonly used algorithms: NetMHC, NetCTLpan and IEDB, and using patient-specific haplotypes HLA-A*02:01/*32:01, HLA-B*40:01:02/*45:01:01, HLAC*06:02/*03:041 (see Rizvi et al., (2015) Science. 348(6230): 124-8). Eight of the antigens identified by ATLAS were not predicted by any of NetMHC, NetCTLpan, or IEDB. (Note that MEW class II epitopes cannot be effectively predicted using currently available algorithms.)
  • FIG. 8 further shows that epitope predictions have a high false positive rate, miss relevant stimulatory neoantigens, and are not able to identify suppressive and/or inhibitory neoantigens.
  • 137 neoantigens predicted by at least one algorithm only 15 (or 11%) were confirmed by ATLAS to effect a CD8 + T cell response in the NSCLC patient.
  • Six of these 15 neoantigens were found to be suppressive and/or inhibitory.
  • ATLAS identified 9+8 stimulatory neoantigens, and 6+3 suppressive and/or inhibitory neoantigens.
  • 47% of stimulatory antigens found by ATLAS were missed by algorithms, and 33% of suppressive and/or inhibitory neoantigens found by ATLAS failed to be identified by algorithms.
  • TAA genes Twenty-six TAA genes (representing 23 unique genes; labelled as “taa1-26” and shown below in Table 5) were cloned into the ATLAS expression vector (Genocea Biosciences), and sequence-verified. Each TAA was recombinantly expressed in E. coli . Protein expression was verified using a surrogate T cell assay (the B3Z hybridoma) which recognizes the C57BL/6 mouse T cell epitope SIINFEKL (SEQ ID NO: 452), which is inserted at the C-terminus of each open reading frame, upstream of the stop codon. Proteins that induced B3Z responses that exceeded 5% of the positive control (the minimal SIINFEKL (SEQ ID NO: 452) epitope pulsed onto antigen presenting cells) were considered expressed.
  • the B3Z hybridoma which recognizes the C57BL/6 mouse T cell epitope SIINFEKL (SEQ ID NO: 452), which is inserted at the C-terminus of each open
  • PBMC peripheral blood mononuclear cell
  • the PBMC were derived from a 50 year-old Caucasian male who had stage IV colorectal cancer.
  • CD8 + T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation.
  • Monocytes were differentiated into dendritic cells (MDDC).
  • FIG. 9 shows representative results for a single CRC patient.
  • TAA coding conventions are shown in Table 5 below.
  • PBMC from 21 CRC patients were screened against a library of 26 known TAAs (shown in Table 5).
  • CD4 + and CD8 + T cells were sorted and non-specifically expanded using anti-CD3 and anti-CD28-coated microbeads, and CD14 + monocytes were differentiated into dendritic cells (MDDC).
  • Library clones were screened in duplicate using 5,000 MDDC and 80,000 T cells, at an E. coli :MDDC ratio of 100:1; 13 replicates of E. coli expressing neon green (NG) were included as negative controls.
  • Assay supernatants were harvested at 24 hours and stored at ⁇ 80° C.
  • Supernatant cytokines were analyzed using Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
  • FIG. 10 shows data for both the CD4 + (grey bars) and CD8 + (black bars) T cell subsets.
  • NG mean negative control
  • ATLAS was applied to screen the entire complement of mutations identified in the tumor of a consented colorectal cancer patient.
  • An ATLAS library was built that expressed 31 mutations unique to this patient.
  • Each clone contained 113 amino acids with the mutation positioned near the center of the construct and sequence-verified.
  • Each clone was recombinantly expressed in E. coli and protein expression was verified using Western Blot.
  • PBMC peripheral blood mononuclear cells
  • CD8 + T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation.
  • CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDC).
  • CD8 + T cells were screened against the 31 library clones, as well as against 2 negative control clones expressing Neon Green (NG).
  • Library clones were screened using 1,500 MDDC and 80,000 T cells, at an E. coli :MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at ⁇ 80° C. Supernatant cytokines were analyzed using a Meso Scale Discovery custom plate.
  • Clones that induced median cytokine responses that exceeded 3 median absolute deviations (MAD) of the median responses to the negative control Neon Green clones (N 2) (indicated by horizontal dotted line in FIG. 11 ) were considered antigens. Clones that reduced median cytokine responses to 3 MAD below the median negative control responses were considered inhibitory and/or suppressive antigens.
  • MAD median absolute deviations
  • FIG. 11 shows results for IFN ⁇ and TNF ⁇ from the patient's CD8 + T cells.
  • the X indicates the median response to the negative controls. Points above the top dotted line indicate neoantigens that stimulate CD8 + T cell responses (black circles). Points below the lower dotted line indicate neoantigens that inhibit and/or suppress CD8 + T cell responses (black squares).
  • 16% of mutations screened (5 of 31) were identified as neoantigens recognized by his/her peripheral blood CD8 + T cells. Additionally, 13% (4 of 31) of mutations screened were identified as inhibitory and/or suppressive neoantigens. There was no overlap of the neoantigens that induced IFN ⁇ compared with TNF ⁇ , but two of the inhibitory neoantigens suppressed both IFN ⁇ and TNF ⁇ .
  • FIGS. 12A and 12B show Venn diagrams representing the limited overlap between CD8 + -specific T cell neoantigens identified by ATLAS and epitope prediction algorithms.
  • MHC class I epitopes were predicted for all screened neoantigens using three commonly used algorithms: NetMHC, NetCTLpan and IEDB, and using patient-specific haplotypes HLA-A*30:02/*32:01, B*18:01/*14:01, C*05:01/*08:02.
  • FIG. 12A and 12B show Venn diagrams representing the limited overlap between CD8 + -specific T cell neoantigens identified by ATLAS and epitope prediction algorithms.
  • MHC class I epitopes were predicted for all screened neoantigens using three commonly used algorithms: NetMHC, NetCTLpan and IEDB, and using patient-specific haplotypes HLA-A*30:02/*32:01, B*18:01/*14:01, C*05
  • FIG. 12A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ⁇ 1 for the mutant peptide but not for wild-type.
  • FIG. 12B represents epitopes predicted to have binding affinity below 500 nM or an IEDB percentile rank of ⁇ 1, irrespective of the wild-type counterpart predictions.
  • none of the neoantigens that were identified by ATLAS were predicted by algorithms, and there were six epitopes predicted that were not identified empirically (100% false positive and 100% false negative rate). For the latter, there was one neoantigen that was identified using ATLAS that was also predicted by all three algorithms used. The remaining four neoantigens were not predicted by any algorithm. There were 26 epitopes predicted that could not be confirmed by ATLAS (therefore the algorithms had a 96% false positive rate and an 80% false negative rate).
  • FIGS. 13A and 13B show Venn diagrams representing the limited overlap between CD8 + -specific T cell inhibitory and/or suppressive neoantigens identified by ATLAS and epitope prediction algorithms. Epitope predictions do not discriminate between stimulatory or inhibitory and/or suppressive antigens, therefore the same MHC predictions used for FIGS. 12A and 12B were applied for the inhibitory and/or suppressive, rather than stimulatory neoantigens.
  • FIG. 13A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ⁇ 1 for the mutant peptide but not for wild-type.
  • FIG. 13A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ⁇ 1 for the mutant peptide but not for wild-
  • ATLASTM was applied to profile T cell recall responses to a set of Tumor Associated Antigens (TAAs) in 34 subjects with various stages of CRC and pre-malignant lesions in an HLA-independent manner. Twenty-six TAA genes (representing 23 unique genes, shown in Table 5) were cloned into the ATLAS expression vector and sequence verified. Each TAA was recombinantly expressed in E. coli , with expression verified using Western Blot analysis.
  • TAAs Tumor Associated Antigens
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • CD4 + and CD8 + T cells were screened against the 26 library clones, as well as against 10 negative control clones expressing Neon Green (NG).
  • Library clones were screened using 1,000-5,000 MDDCs and 80,000 T cells, at an E. coli :MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at ⁇ 80° C. Supernatant cytokines levels were analyzed using a Meso Scale Discovery custom plate.
  • FIG. 14 shows response profiles to 25 CRC-associated TAAs across CRC patients.
  • CD4 + and CD8 + T cells from CRC patients across all stages of disease were profiled for responses to 25 TAAs, using TNF- ⁇ and IFN- ⁇ secretion as an indicator for a recall response to a putative antigen.
  • Distributions of normalized cytokine concentrations released in response to each antigen are shown, each row represents one antigen.
  • Dashed vertical lines indicate 2 MADs from median cytokine release in response to the NG negative control antigen.
  • Positive values indicated by a shift toward the right side of the plot, indicate stimulatory T cell recall responses.
  • Negative values, indicated by a shift toward the left side of the plot indicate inhibitory and/or suppressive T cell recall responses.
  • FIGS. 15A and 15B shows the high frequency of T cell responses to three TAAs not previously identified by algorithm. Response rates in individuals with CRC to three ATLAS-identified TAAs in comparison to three TAAs that are or were in clinical development as a therapeutic vaccine.
  • FIG. 15A shows response rate of CD4 + and CD8 + T cells for HPSE1 and HPSE2, in comparison to MUC1, MAGEA3, and TP53.
  • FIG. 15B shows response rate of CD8 + T cells for HPSE1, HPSE2 and SMAD4, in comparison to MUC1, MAGEA3, and TP53. Stimulatory (top panels) and inhibitory and/or suppressive (bottom panels) T cell recall responses are shown.
  • TNF- ⁇ levels were detected across the same groups (not shown).
  • T cell responses to a subset of TAAs HPSE1, HPSE2, SMAD4 in individuals with pre-malignant adenomatous polyps were similar to those in CRC patients and clearly distinguishable from the rare responses in healthy individuals. This pattern was not observed for responses to TAAs currently or previously investigated as therapeutic vaccines (MUC1, TP53, MAGEA3).
  • ATLAS Tumor Associated Antigens
  • PBMC peripheral blood mononuclear cells
  • CD4 + and CD8 + T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation.
  • CD14 + monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDCs).
  • CD4 + and CD8 + T cells were screened against the 76 library clones, as well as against 10 negative control clones expressing Neon Green (NG).
  • Library clones were screened using 1,000-5,000 MDDCs and 80,000 T cells, at an E. coli :MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at ⁇ 80° C. Supernatant cytokines levels were analyzed using a Meso Scale Discovery custom plate.
  • Clones that induced median cytokine responses that exceeded 2 median absolute deviations (MADs) of the median responses to the negative control Neon Green clones (N 10) (indicated by a horizontal dotted line in FIG. 18 and FIG. 19 ) were considered antigens. Clones that reduced median cytokine responses to two MADs below the median negative control responses were considered inhibitory and/or suppressive antigens.
  • MADs median absolute deviations
  • FIG. 18 shows an exemplary empirical determination of T cell responses to profiled TAAs.
  • Exemplary data is shown for a single lung cancer patient.
  • T cell responses were reported as natural log concentrations back-calculated from the MSD standard curve and normalized to the patient's response to a negative control protein.
  • a stimulatory response was defined as a TAA with a median concentration greater than two MADs above the median of the negative control replicates. This threshold is shown as the upper dashed horizontal line, and stimulatory responses are shown as filled circles.
  • An inhibitory and/or suppressive response was defined as a TAA with a median concentration greater than two MADs of the negative control replicates below the median of the negative control replicates. This threshold is shown as the lower dashed horizontal line, and inhibitory and/or suppressive responses are shown as filled triangles.
  • FIG. 19 shows frequent CD4 + T cell responses to novel TAAs compared to previously described TAAs.
  • Each point represents a patient's response to that TAA, normalized to the patient's response to an irrelevant negative control protein.
  • Stimulatory responses those that fall above the 2 ⁇ MAD cutoff indicated by the upper horizontal dotted line, are colored black. Both the median normalized concentration and the proportion of stimulatory responses to these two TAAs were higher than those of the three other TAAs.
  • CD8+ responses to these five TAAs were more similar across patients (not shown).
  • FIG. 20 shows that lung cancer patients develop CD4 + and CD8 + T cell responses to a broad range of TAAs.
  • stimulatory CD4 + and/or CD8 + T cell responses were observed in at least one individual to a clear majority of the 76 profiled TAAs.
  • the percent of patients that developed a stimulatory T cell response to each TAA is shown separately for CD4 + (grey bars) and CD8 + (black bars) T cells.
  • IFN- ⁇ responses are displayed in the top two panels, and TNF- ⁇ responses are displayed in the bottom two panels.
  • Antigens to which patients developed both a CD4 + and a CD8 + T cell response were differentiated from antigens to which patients developed either a CD4 + or a CD8 + T cell response (right panels).
  • FIG. 21 shows that inhibitory and/or suppressive T cell responses were detected in most profiled TAAs. Inhibitory and/or suppressive T cell responses to TAAs were observed frequently across the profiled lung cancer patients. For each profiled TAA, the percent of patients that developed an inhibitory and/or suppressive T cell response, defined as a response that is two MADs lower than the response to the negative control protein, are shown for CD4 + (white bars) and CD8 + (grey bars) T cells. IFN- ⁇ responses are displayed in the top two panels, and TNF- ⁇ responses are displayed in the bottom two panels. Antigens to which patients developed both a CD4 + and a CD8 + T cell response (left panels) were differentiated from antigens to which patients developed either a CD4 + or a CD8 + T cell response (right panels).
  • ATLAS was applied to characterize and profile pre-existing T cell responses to tumor specific mutations in a diverse set of cancer patients.
  • Tumor biopsy and normal tissue samples were collected from 19 consenting patients.
  • Whole exome and RNA sequencing of the tumor sample and whole exome sequencing of the matched normal sample identified mutations which are unique to the tumor and not present in the germline of the patient.
  • Each somatic protein altering mutation was expressed as individual clones in the ATLAS expression vector and sequence verified. Each clone was recombinantly expressed in E. coli , with expression verified using Western Blot analysis.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs Frozen peripheral blood mononuclear cells
  • CD4 + and CD8 + T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation.
  • CD14 + monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into myeloid derived dendritic cells (MDDCs).
  • MDDCs myeloid derived dendritic cells
  • CD4 + and CD8 + T cells were screened against the individuals' specific library clones, as well as against multiple negative control clones expressing Neon Green (NG).
  • Library clones were screened using 1,000-5,000 MDDCs and 80,000 T cells, at an E. coli :MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at ⁇ 80° C. Supernatant cytokines levels were analyzed using a Meso Scale Discovery custom plate.
  • MADs median absolute deviations
  • FIG. 22 shows an exemplary neoantigen screen with ATLAS identifying patient-specific CD4 + and CD8 + T cell responses.
  • displayed are the CD4+ and CD8+ T cell responses observed in response to each candidate neoantigen.
  • Each dot represents a technical replicate.
  • Horizontal dotted lines indicate the cutoffs used to define stimulatory neoantigens and inhibitory and/or suppressive neoantigens at +3 and ⁇ 3 Median Absolute Deviations (MADs), respectively.
  • MADs Median Absolute Deviations
  • FIGS. 23A, 23B, 23C, and 23D show that an algorithm predicting MHC Class I binding did not accurately predict CD8 + T cell responses or type of response.
  • the diagrams compare MHC class I algorithm-based binding predictions (NetMHCpan predictions with binding affinity cutoff of ⁇ 500 nM) and T cell responses observed in ATLAS across 11 initial subjects and across all 19 subjects.
  • FIGS. 23A and 23C show the total numbers and overlap of neoantigens predicted by algorithm and observed in ATLAS for the 11 initial subjects and for all 19 subjects, respectively.
  • FIGS. 23A, 23B, 23C, and 23D show that an algorithm predicting MHC Class I binding did not accurately predict CD8 + T cell responses or type of response.
  • the diagrams compare MHC class I algorithm-based binding predictions (NetMHCpan predictions with binding affinity cutoff of ⁇ 500 nM) and T cell responses observed in ATLAS across 11 initial subjects and across all 19 subjects.
  • FIGS. 23A and 23C show the total numbers and overlap of neo
  • FIGS. 24A and 24B show that CD8 + T cell responses identified by ATLAS to candidate stimulatory neoantigens were not enriched for any mutation type.
  • mutation types for the 11 initial subjects were defined as missense, in-frame, or frameshift.
  • mutation types for all 19 subjects were defined as short variant (a combination of missense and in-frame mutations resulting in 1-2 amino acid changes relative to wild-type gene sequence) and neoORF (a combination of frameshift and loss-of-stop-codon mutations resulting in 3 or more amino acid changes relative to wild-type gene sequence).
  • candidate inhibitory and/or suppressive neoantigens were somewhat more frequently associated with missense or short variant mutations.
  • FIG. 25A shows results for the 11 initial subjects.
  • FIG. 25B shows results for all 19 subjects.
  • FIGS. 26A and 26B show that detection of a mutation in RNA did not predict whether the candidate stimulatory or inhibitory/suppressive antigen has a recall response in CD8 + T cells.
  • RNA-seq was performed on the tumor material. Somatic mutations were identified via whole exome sequencing, and the RNA-seq data was interrogated for the presence or absence of mutations identified in DNA.
  • FIG. 26A shows results for 8 of the 11 initial subjects.
  • FIG. 26B shows results for all 19 subjects.
  • FIGS. 27A and 27B show that CD8+ T cell responses identified by ATLAS to candidate neoantigens did not correlate with gene expression.
  • RNA-seq was performed on the tumor material; quantitative gene expression values were calculated for each gene harboring a candidate neoantigen and compared to normalized cytokine measurements.
  • FIG. 27A shows results for 10 of the 11 initial subjects.
  • FIG. 27B shows results for all 19 subjects.
  • ATLAS was applied to screen the entire complement of mutations identified in the tumor of a consented pancreatic cancer patient.
  • An ATLAS library was built that expressed 22 mutations unique to this patient.
  • Each clone contained 113 amino acids with the mutation positioned near the center of the construct and sequence-verified.
  • Each clone was recombinantly expressed in E. coli and protein expression was verified using Western Blot.
  • PBMC peripheral blood mononuclear cells
  • CD8 + T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation.
  • CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDC).
  • CD8 + T cells were screened against the 22 library clones, as well as against a negative control clones expressing Neon Green (NG).
  • Library clones were screened using 5,000 MDDC and 80,000 T cells, at an E. coli :MDDC ratio of 333:1. After 19.5 h incubation, assay supernatants were harvested and stored at ⁇ 80° C. Supernatant cytokines CM-CSF, IFN ⁇ , IL-10, MIF, TNF ⁇ , and TRAIL were analyzed using a Meso Scale Discovery custom plate.
  • FIG. 28 shows the different CM-CSF, IFN ⁇ , IL-10, MIF, TNF ⁇ , and TRAIL response profiles elicited by six representative neoantigens in a screen of CD8 + T cells from the patient.
  • Each panel corresponds to one neoantigen (denoted G-3618, G-3624, G-3620, G-3627, G-3617, and G-3632).
  • the horizontal line in each panel indicates the median response to the negative controls. Bars above the horizontal line indicate stimulation of cytokine secretion. Bars below the horizontal line indicate inhibition and/or suppression of cytokine secretion.
  • the panels illustrate the different cytokine responses elicited by each neoantigen.
  • PBMC from eight cancer patients (seven lung cancer, one colorectal cancer) and 13 healthy donors were screened in duplicate against VEGF, a known TAA.
  • CD8 + T cells were sorted and non-specifically expanded using anti-CD3 and anti-CD28-coated microbeads, and CD14 + monocytes were differentiated into dendritic cells (MDDC).
  • Library clones were screened in duplicate using 5,000 MDDC and 80,000 T cells, at an E. coli :MDDC ratio of 100:1; replicates of E. coli expressing neon green (NG) were included as negative controls.
  • Assay supernatants were harvested at 24 hr and stored at ⁇ 80° C.
  • Supernatant cytokines were analyzed using Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
  • FIG. 29 shows CD8 + T cell data for healthy donors (black bars) and cancer patients (white bars). The median log cytokine response normalized to neon green are indicated for each subject cohort. When analyzed by IFN ⁇ secretion, there was a large inhibitory response in the healthy donor cohort, that greatly exceeded the inhibitory responses in the cancer patient cohort. Conversely, there was a greater median inhibitory response in the cancer cohort when TNF ⁇ secretion was considered.
  • PBMCs from healthy donors are enriched using standard protocols. Washed PBMCs are resuspended in supplemented RPMI-1640 medium. 100 ⁇ L cells (2 ⁇ 10 6 cell/mL) are added into each well of a 96-well flat-bottom assay plate. Overlapping peptides corresponding to TAAs HPSE1, HPSE2, SMAD4, MUC1, MAGEA3, and TP53 were added to cultures at a final concentration of 50 ⁇ g/mL. Cultures are incubated for 5 days, the peptide-containing medium removed, then cultures provided with human IL-2 (10 U/mL) for 11 days, with IL-2-containing medium being replenished every 3 days.
  • the incubation time of 5 days with peptide plus 11 days with IL-2 constitutes one cycle.
  • Primary cultures are subsequently restimulated with the same peptides (50 ng/mL) on day 16 to begin the next cycle.
  • Irradiated (4000 rad) autologous peripheral blood mononuclear cells (5 ⁇ 10 5 ) are added in a volume of 50 ⁇ L in complete medium as APCs.
  • An ELISPOT is performed on an aliquot of cells at the end of each cycle to observe de novo responses to the peptides.

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Abstract

Methods and compositions for identifying tumor antigens of human lymphocytes, and for identifying subjects for cancer therapy, are provided herein.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application is a divisional U.S. application Ser. No. 15/927,067, filed Mar. 20, 2018, now U.S. Pat. No. 10,859,566, which claims the benefit of U.S. Provisional Application No. 62/583,233, filed Nov. 8, 2017, U.S. Provisional Application No. 62/484,258, filed Apr. 11, 2017 and U.S. Provisional Application No. 62/473,899, filed Mar. 20, 2017, the contents of each of which are hereby incorporated by reference herein in their entirety.
    • SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 22, 2021, is named 2007781-0274_SL.txt and is 2,106,037 bytes in size.
    • BACKGROUND
  • Cancer is characterized by proliferation of abnormal cells. Many treatments include costly and painful surgeries and chemotherapies. Although there is a growing interest in cancer therapies that target cancerous cells using a patient's own immune system, such therapies have had limited success.
    • SUMMARY
  • The present invention features, inter alia, methods of identifying tumor antigens and potential tumor antigens of human lymphocytes, methods of selecting tumor antigens and potential tumor antigens, as well as compositions including the tumor antigens and potential tumor antigens, methods of making such compositions, and methods of using the tumor antigens and potential tumor antigens. The invention also features methods of evaluating an immune response in a cancer subject, e.g., for identifying and/or selecting a cancer subject for initiation, continuation, modification, and/or discontinuation of a cancer therapy
  • Accordingly, in one aspect the disclosure features a method of obtaining or generating a subject response profile. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads of the library with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; and e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate a subject response profile.
  • In some embodiments, the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of obtaining or generating a target response profile. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads of the library with antigen presenting cells (APCs) from a subject who exhibits or previously exhibited a response to cancer, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; and e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate a target response profile.
  • In some embodiments, the subject exhibits or previously exhibited at least one beneficial response to cancer. In some embodiments, the beneficial response comprises a positive clinical response, e.g., one or more positive clinical endpoints, to a cancer therapy or combination of therapies. In some embodiments, the beneficial response comprises a spontaneous response to a cancer. In some embodiments, the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer. In some embodiments, the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years). In some embodiments, the beneficial response comprises a positive cancer prognosis. In some embodiments, the beneficial response comprises a lack of measurable toxic responses or side effects to a cancer therapy or combination of therapies.
  • In some embodiments, the subject exhibits or previously exhibited at least one deleterious or non-beneficial response to cancer. In some embodiments, the deleterious response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies. In some embodiments, the deleterious response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer. In some embodiments, the deleterious response comprises at least one relapse, recurrence, and/or metastasis of a cancer. In some embodiments, the deleterious response comprises a negative cancer prognosis. In some embodiments, the deleterious response comprises one or more toxic responses or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the library used to obtain the target response profile is the same library used to obtain a subject response profile.
  • In some embodiments, the method further comprises the step of repeating steps a) through e) with antigen presenting cells (APCs) and/or lymphocytes from additional subjects, to obtain a population-based or composite target response profile.
  • In some embodiments, the target response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of identifying a subject as a candidate for cancer therapy. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate a subject response profile; and f) comparing the subject response profile to a target response profile to select the subject as a candidate subject for initiation, continuation, modification, discontinuation or non-initiation of a cancer therapy. In some embodiments, the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the method further comprises generating the target response profile by a method comprising: g) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; h) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; i) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and j) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate the target response profile. In some embodiments, the target response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer. In some embodiments, the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies. In some embodiments, the beneficial response comprises a spontaneous response to a cancer. In some embodiments, the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer. In some embodiments, the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years). In some embodiments, the beneficial response comprises a positive cancer prognosis. In some embodiments, the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies. In some embodiments, the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises selecting the candidate subject for initiation of a cancer therapy or combination of cancer therapies. In some embodiments, the method further comprises selecting the candidate subject for continuation of a cancer therapy or combination of cancer therapies. In some embodiments, the method comprises selecting the subject as a candidate subject (i) if the subject response profile is similar to the target response profile from a target subject who exhibits or previously exhibited one or more beneficial responses to the cancer therapy or combination, and/or (ii) if the subject response profile is dissimilar to the target response profile from a target subject who exhibits or previously exhibited one or more deleterious responses to the cancer therapy or combination. In some embodiments, the method further comprises administering the cancer therapy or combination of cancer therapies to the candidate subject.
  • In some embodiments, the method further comprises selecting the candidate subject for modification of a cancer therapy. In some embodiments, the method further comprises selecting the candidate subject for discontinuation or non-initiation of a cancer therapy. In some embodiments, the method further comprises selecting the subject as a candidate subject for modification, discontinuation, and/or non-initiation of a cancer therapy (i) if the subject response profile is similar to the target response profile from a target subject who exhibits or previously exhibited one or more deleterious responses to the cancer therapy, and/or (ii) if the subject response profile is dissimilar to the target response profile from a target subject who exhibits or previously exhibited one or more beneficial responses to the cancer therapy. In some embodiments, the method further comprises modifying the cancer therapy administered to the candidate subject. In some embodiments, the method further comprises discontinuing or not initiating the cancer therapy to the candidate subject.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of selecting tumor antigens. In some embodiments, the, method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level) e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate a subject response profile; f) comparing the subject response profile to a target response profile; and g) selecting one or more tumor antigens based on the comparison. In some embodiments, the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the method further comprises generating the target response profile by a method comprising: h) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; i) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; j) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and k) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate the target response profile. In some embodiments, the target response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited one or more beneficial response to cancer. In some embodiments, the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies. In some embodiments, the beneficial response comprises a spontaneous response to a cancer. In some embodiments, the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer. In some embodiments, the beneficial response comprises a relapse, recurrence, and/or metastasis of a cancer e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years). In some embodiments, the beneficial response comprises a positive cancer prognosis. In some embodiments, the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious or non-beneficial response to cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies. In some embodiments, the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises selecting (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with a beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with deleterious or not beneficial responses to cancer. In some embodiments, the method further comprises administering to the subject an immunogenic composition comprising one or more of the selected antigens or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises selecting (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with deleterious or not beneficial responses to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with beneficial responses to cancer. In some embodiments, the method further comprises administering to the subject an immunogenic composition that does not comprise one or more of the selected antigens or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate a subject response profile; f) comparing the subject response profile to a target response profile; g) selecting one or more tumor antigens based on the comparison; and h) administering to the subject an immunogenic composition comprising one or more of the selected antigens or immunogenic fragment thereof. In some embodiments, the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the method further comprises generating the target response profile by a method comprising: i) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; j) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; k) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and l) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate the target response profile. In some embodiments, the target response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer. In some embodiments, the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies. In some embodiments, the beneficial response comprises a spontaneous response to a cancer. In some embodiments, the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer. In some embodiments, the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years). In some embodiments, the beneficial response comprises a positive cancer prognosis. In some embodiments, the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises selecting and administering to the subject (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with one or more beneficial responses to cancer, and/or (i) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with one or more deleterious or not beneficial responses to cancer.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate a subject response profile; f) comparing the subject response profile to a target response profile; g) selecting one or more tumor antigens based on the comparison; and h) administering to the subject an immunogenic composition that does not comprise one or more of the selected antigens or immunogenic fragment thereof. In some embodiments, the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the method further comprises generating the target response profile by a method comprising: i) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a target subject, wherein the APCs internalize the bacterial cells or beads; j) contacting the APCs with lymphocytes from the target subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; k) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; and l) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain or generate the target response profile. In some embodiments, the target response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies. In some embodiments, the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises selecting one or more tumor antigens that increase expression or secretion of immune mediators associated with deleterious or not beneficial responses to cancer, and/or one or more tumor antigens that inhibit and/or suppress expression or secretion of immune mediators associated with beneficial responses to cancer.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of selecting tumor antigens. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by one or more tumor antigens presented by one or more APCs by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain a subject response profile; and f) selecting from among the identified tumor antigens one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or selecting one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer.
  • In some embodiments, the method further comprises comparing the subject response profile to a target response profile, e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer. In some embodiments, the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies. In some embodiments, the beneficial response comprises a spontaneous response to a cancer. In some embodiments, the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer. In some embodiments, the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years). In some embodiments, the beneficial response comprises a positive cancer prognosis. In some embodiments, the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises administering to the subject an immunogenic composition comprising one or more of the selected antigens or immunogenic fragments thereof.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of selecting tumor antigens. In some embodiments, the method comprises a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by one or more tumor antigens presented by one or more APCs by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain a subject response profile; and f) selecting from among the identified tumor antigens (i) one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer.
  • In some embodiments, the method further comprises comparing the subject response profile to a target response profile, e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies. In some embodiments, the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises administering to the subject an immunogenic composition that does not comprise one or more of the selected antigens or immunogenic fragments thereof.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators, to obtain a subject response profile; f) selecting from among the identified tumor antigens (i) one or more antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one deleterious or non-beneficial response to cancer; and g) administering to the subject an immunogenic composition comprising one or more of the selected antigens or immunogenic fragment thereof.
  • In some embodiments, the method further comprises comparing the subject response profile to a target response profile, e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison, prior to administration of the immunogenic composition.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited at least one beneficial response to cancer. In some embodiments, the beneficial response comprises a positive clinical response to a cancer therapy or combination of therapies. In some embodiments, the beneficial response comprises a spontaneous response to a cancer. In some embodiments, the beneficial response comprises clearance of a cancer, e.g., a level of one or more clinical measures associated with clearance of a cancer. In some embodiments, the beneficial response comprises a lack of a relapse, recurrence, and/or metastasis of a cancer, e.g., over a defined period of time (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 years). In some embodiments, the beneficial response comprises a positive cancer prognosis. In some embodiments, the beneficial response comprises a lack of one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control). of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, to obtain a subject response profile; f) comparing the subject response profile to a target response profile, e.g., a target response profile generated using a method described herein; g) selecting from among the identified tumor antigens (i) one or more antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer; and h) administering to the subject an immunogenic composition that does not comprise one or more of the selected antigens or immunogenic fragment thereof.
  • In some embodiments, the method further comprises comparing the subject response profile to a target response profile e.g., a target response profile generated using a method described herein, and selecting one or more tumor antigens based on the comparison.
  • In some embodiments, the target response profile is from one or more target subjects who exhibit or previously exhibited one or more deleterious and/or non-beneficial response to cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative clinical response and/or a failure to respond, to a cancer therapy or combination of therapies. In some embodiments, the deleterious and/or non-beneficial response comprises a lack of clearance of a cancer, e.g., a level of one or more clinical measures associated with lack of clearance of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises at least one relapse, recurrence, and/or metastasis of a cancer. In some embodiments, the deleterious and/or non-beneficial response comprises a negative cancer prognosis. In some embodiments, the deleterious and/or non-beneficial response comprises one or more toxic responses and/or side effects (e.g., one or more measurable toxic responses and/or side effects) to a cancer therapy or combination of therapies.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of identifying tumor antigens. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; and e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, wherein stimulation, inhibition and/or suppression indicate that the polypeptide is a tumor antigen.
  • In another aspect, the disclosure features a method of selecting tumor antigens. In some embodiments, the method comprises: a) providing a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, wherein stimulation, inhibition and/or suppression indicate that the polypeptide is a tumor antigen; and f) selecting from among the identified tumor antigens (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer.
  • In some embodiments, the method further comprises selecting from among the identified polypeptides one of more polypeptides that have a minimal effect on level of expression and/or secretion of one of more immune mediators.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of selecting potential tumor antigens. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, and identifying one or more polypeptides that stimulate, inhibit, and/or suppress as a tumor antigen; and f) selecting from among the identified polypeptides one or more polypeptides that have a minimal effect on level of expression and/or secretion of one or more immune mediators.
  • In some embodiments, the method further comprises repeating steps b) through e), or steps c) through e), with lymphocytes from the subject that have undergone one or more previous rounds of exposure to APCs.
  • In some embodiments, the method further comprises selecting from among the identified tumor antigens (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial responses to cancer.
  • In some embodiments, the method further comprises administering to the subject an immunogenic composition comprising one or more of the selected tumor antigens or selected polypeptides, or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject an immunogenic composition comprising a combination of one or more of the selected tumor antigens and selected polypeptides, or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of selecting tumor antigens. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, and identifying one or more polypeptides that stimulate, inhibit, and/or suppress as a tumor antigen; and f) selecting from among the identified tumor antigens (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer.
  • In some embodiments, the method further comprises administering to the subject an immunogenic composition that does not comprise one or more of the selected tumor antigens or immunogenic fragments thereof. In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In some embodiments, the APCs are human APCs isolated from the subject; and/or the bacterial cells further comprise a cytolysin polypeptide; and/or the cytolysin polypeptide is listeriolysin O (LLO); and/or the APCs are provided in an array, and/or the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen; and/or the APCs and lymphocytes are isolated from peripheral blood; and/or the APCs comprise immortalized cells; and/or the lymphocytes are derived from a cancer or tumor.
  • In some embodiments, the tumor antigens comprise full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding mutations, splice variants, or translocations present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoded by a virus or other infectious agent present in a cancer or tumor; and/or the tumor antigens comprise full length polypeptides encoding autoantigens associated with a cancer or tumor; and/or the tumor antigens comprise polypeptides that are fragments of full length polypeptides encoding autoantigens associated with a cancer or tumor.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, and identifying a polypeptide that stimulates, inhibits and/or suppresses as a tumor antigen; f) selecting from among the identified tumor antigens (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial responses to cancer; and g) administering to the subject an immunogenic composition comprising one or more of the selected antigens or immunogenic fragment thereof.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, wherein stimulation, inhibition and/or suppression indicate that the polypeptide is a tumor antigen; f) selecting from among the identified polypeptides one or more polypeptides that have a minimal effect on level of expression and/or secretion of one or more immune mediators; and g) administering to the subject an immunogenic composition comprising one or more of the selected polypeptides or immunogenic fragment thereof.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control), of expression and/or secretion of one or more immune mediators; e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, wherein stimulation, inhibition and/or suppression indicate that the polypeptide is a tumor antigen; f) selecting from among the identified tumor antigens and polypeptides (i) one or more polypeptides that have a minimal effect on level of expression and/or secretion of one or more immune mediators, (ii) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer; and/or (iii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer; and g) administering to the subject an immunogenic composition comprising one or more of the selected tumor antigens and polypeptides, or immunogenic fragments thereof.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject. In some embodiments, the method comprises: a) obtaining, providing, or generating a library comprising bacterial cells or beads, wherein each bacterial cell or bead of the library comprises a different heterologous polypeptide comprising one or more mutations, splice variants, or translocations expressed in a cancer or tumor cell of a subject; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a polypeptide presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more polypeptides presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more polypeptides that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, and identifying a polypeptide that stimulates, inhibits and/or suppresses as a tumor antigen; f) selecting from among the identified tumor antigens (i) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer; and g) administering to the subject an immunogenic composition that does not comprise one or more of the selected antigens or immunogenic fragment thereof.
  • In some embodiments, the method further comprises administering to the subject a cancer therapy or combination of therapies.
  • In any of the aspects described herein, the plurality of tumor antigens comprises at least 1, 3, 5, 10, 15, 20, 25, 30, 50, 100, 150, 250, 500, 750, 1000 or more different tumor antigens, or portions thereof; and/or determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens comprises measuring a level of one or more immune mediators; and/or the one or more immune mediators are selected from the group consisting of cytokines, soluble mediators, and cell surface markers expressed by the lymphocytes; and/or the one or more immune mediators are cytokines; and/or the one or more cytokines are selected from the group consisting of TRAIL, IFN-gamma, IL-12p70, IL-2, TNF-alpha, MIP1-alpha, MIP1-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3-alpha, and fractalkine; and/or the one or more immune mediators are soluble mediators; and/or the one or more soluble mediators are selected from the group consisting of granzyme A, granzyme B, sFas, sFasL, perform, and granulysin; and/or the one or more immune mediators are cell surface markers; and/or the one or more cell surface markers are selected from the group consisting of CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD1 (CD279), FoxP3, TIGIT, CD160, BTLA, 2B4 (CD244), and KLRG1; and/or the lymphocytes comprise CD4+ T cells; and/or the lymphocytes comprise CD8+ T cells; and/or the lymphocytes comprise NKT cells, gamma-delta T cells, or NK cells; and/or the lymphocytes comprise any combination of CD4+ T cells, CD8+ T cells, NKT cells, gamma-delta T cells, and NK cells; and/or lymphocyte activation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher or lower than a control level; and/or lymphocyte activation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least one, two, or three standard deviations greater or lower than the mean of a control level; and/or lymphocyte activating is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control; and/or lymphocyte non-responsiveness is determined by assessing a level of one or more expressed or secreted immune mediators that is within 5%, 10%, 15%, or 20% of a control level; and/or lymphocyte non-responsiveness is determined by assessing a level of one or more expressed or secreted immune mediators that is less than one or two standard deviation higher or lower than the mean of a control level; and/or lymphocyte non-responsiveness is determined by assessing a level of one or more expressed or secreted immune mediators that is less than one or two median absolute deviation (MAD) higher or lower than a median response level to a control; and/or the subject response profile comprises one or more different tumor antigens that increase level of expression and/or secretion of one or more immune mediators; and/or the subject response profile comprises one or more different tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators; and/or the subject response profile comprises one or more different tumor antigens that have a minimal effect on level of expression and/or secretion of one or more immune mediators; and/or the subject response profile comprises a combination of one or more different tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators; and/or the target response profile comprises one or more different tumor antigens that increase level of expression and/or secretion of one or more immune mediators; and/or the target response profile comprises one or more different tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators; and/or the target response profile comprises one or more different tumor antigens that have a minimal effect on level of expression and/or secretion of one or more immune mediators; and/or the target response profile comprises a combination of one or more different tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators; and/or the target response profile comprises an average number of different tumor antigens that increase level of expression and/or secretion of one or more immune mediators, from a population of subjects who respond clinically to the cancer therapy, or from subjects who fail to respond clinically to the cancer therapy; and/or the target response profile comprises an average number of different tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators, from a population of subjects who respond clinically to the cancer therapy, or from subjects who fail to respond clinically to the cancer therapy; and/or the target response profile comprises an average number of different tumor antigens that have a minimal effect on level of expression and/or secretion of one or more immune mediators, from a population of subjects who respond clinically to the cancer therapy, or from subjects who fail to respond clinically to the cancer therapy; and/or the target response profile comprises a combination of different tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on level of expression and/or secretion of one or more immune mediators, from a population of subjects who respond clinically to the cancer therapy, or from a population of subjects who fail to respond clinically to the cancer therapy; and or the subject response profile is similar to the target response profile if the number of tumor antigens of the subject response profile differs by no more than 1, 2, 3, 4, 5, or 10 from the number of antigens of the target response profile; and/or the subject response profile comprises the number of different tumor antigens for each of a plurality of cytokines expressed and/or secreted by activated and/or non-responsive lymphocytes; and/or the target response profile comprises the number of antigens for each of the corresponding plurality of cytokines; and/or the target response profile comprises an average number of antigens for each of the corresponding plurality of cytokines expressed and/or secreted by activated and/or non-responsive lymphocytes from a population of subjects who respond clinically to the cancer therapy; and/or the target response profile comprises an average number of antigens for each of the corresponding plurality of cytokines expressed and/or secreted by activated and/or non-responsive lymphocytes from a population of subjects who fail to respond clinically to the cancer therapy; and/or the target response profile comprises a combination of antigens for each of the corresponding plurality of cytokines expressed and/or secreted by activated and/or non-responsive lymphocytes from a population of subjects who respond clinically to the cancer therapy, or from a population of subjects who fail to respond clinically to the cancer therapy; and/or the subject response profile is similar to the target response profile if the number of tumor antigens for at least two of the plurality of cytokines of the subject response profile differs by no more than 1, 2, 3, 4, 5, or 10 from the number of antigens for the corresponding plurality of cytokines of the target response profile; and/or a subject exhibits at least one measure or indication of clinical responsiveness to the cancer therapy; and/or a subject exhibits at least one measure or indication of failure of clinical responsiveness to the cancer therapy; and/or the cancer therapy comprises immune checkpoint blockade therapy; and/or the immune checkpoint blockade therapy comprises administration of pembrolizumab, nivolumab, ipilimumab, atezolizumab, avelumab, durvalumab, tremelimumab, or cemiplimab; and/or the immune checkpoint blockade therapy comprises administration of two or more immune checkpoint inhibitors; and/or the cancer therapy comprises immune suppression blockade therapy; and/or the immune suppression blockade therapy comprises administration of Vista (B7-H5, v-domain Ig suppressor of T cell activation) inhibitors, Lag-3 (lymphocyte-activation gene 3, CD223) inhibitors, IDO (indolemamine-pyrrole-2,3,-dioxygenase-1,2) inhibitors, or KIR receptor family (killer cell immunoglobulin-like receptor) inhibitors, CD47 inhibitors, or Tigit (T cell immunoreceptor with Ig and ITIM domain) inhibitors; and/or the immune suppression blockade therapy comprises administration of two or more immune suppression inhibitors; and/or the cancer therapy comprises immune activation therapy; and/or the immune activation therapy comprises administration of CD40 agonists, GITR (glucocorticoid-induced TNF-R-related protein, CD357) agonists, OX40 (CD134) agonists, 4-1BB (CD137) agonists, ICOS (inducible T cell stimulator, CD278) agonists, IL-2 (interleukin 2) agonists, or interferon agonists; and/or immune activation comprises administration of two or more immune activators; and/or the cancer therapy comprises adjuvant therapy; and/or the adjuvant therapy comprises administration of a TLR agonist (e.g., CpG or Poly I:C), STING agonist, non-specific stimulus of innate immunity, dendritic cells, GM-CSF, IL-12, IL-7, Flt-3, or other cytokines; and/or the cancer therapy comprises oncolytic virus therapy; and/or the oncolytic viral therapy comprises administration of talimogene leherparepvec; and/or the cancer therapy comprises administration of one or more chemotherapeutic agents; and/or the cancer therapy comprises radiation; and/or the cancer therapy comprises surgical excision; and/or the cancer therapy comprises cell-based therapy; and/or the cell-based therapy comprises administration of dendritic cells, chimeric antigen receptor T (CAR-T) cells, T cell receptor-transduced cells, tumor infiltrating lymphocytes (TIL), or natural killer (NK) cells; and/or the cancer therapy comprises localized hyperthermia or hypothermia; and/or the cancer therapy comprises administration of one or more anti-tumor antibodies; and/or the anti-tumor antibodies comprise bi-specific antibodies; and/or the cancer therapy comprises administration of one or more anti-angiogenic agents; and/or the cancer therapy comprises any combination of immune checkpoint blockade, immune suppression blockade, immune activation, adjuvant, oncolytic virus, chemotherapeutic, radiation, surgical, cell-based, hyperthermia, hypothermia, anti-tumor antibody, and anti-angiogenic therapies.
  • In another aspect, the disclosure features a method of inducing an immune response in a subject with one or more selected antigens, the method comprising: a) obtaining, providing or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a first subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the first subject, under conditions suitable for stimulation or inhibition and/or suppression of lymphocytes by a tumor antigen presented by one or more APCs; d) identifying one or more stimulatory tumor antigens that stimulate lymphocytes and identifying one or more non-stimulatory tumor antigens that do not stimulate lymphocytes, to produce a subject response profile; e) comparing the subject response profile to a target response profile, wherein the target response profile is from a second subject who responds clinically to a cancer therapy, and wherein the target response profile comprises one or more identified stimulatory tumor antigens that stimulate lymphocytes and comprises one or more identified non-stimulatory tumor antigens that do not stimulate lymphocytes; f) selecting one or more antigens, wherein the one or more antigens are identified as non-stimulatory in the subject response profile and the same one or more antigens are identified as stimulatory in the target response profile; and g) administering to the first subject an immunogenic composition comprising one or more of the selected antigens.
  • In some embodiments, the method further comprises administering a cancer therapy to the subject. In some embodiments, the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
  • In another aspect, the disclosure features an immunogenic composition of the invention, comprising one or more antigens of the target response profile obtained or generated according to any of the methods described herein.
  • In another aspect, the disclosure features an immunogenic composition of the invention, comprising one or more antigens selected according to any of the methods described herein.
  • In another aspect, the disclosure features an immunogenic composition comprising (i) one or more heparanase polypeptides or immunogenic fragments thereof and (ii) a SMAD4 polypeptide or immunogenic fragment thereof.
  • In some embodiments, the one or more heparanase polypeptides or fragments and the SMAD4 polypeptide or fragment are each 8-29 amino acids in length. In some embodiments, the heparanase polypeptides comprise the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the SMAD4 polypeptide comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the one or more immunogenic fragments consist of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total number of amino acids of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, the one or more immunogenic fragments consist of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 lacking about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some embodiments, the one or more heparanse polypeptides comprise an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the SMAD4 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:8.
  • In another aspect, the disclosure features an immunogenic composition comprising a heparanase isoform 1 polypeptide or immunogenic fragment, a heparanase isoform 2 polypeptide or immunogenic fragment, and a SMAD4 polypeptide or immunogenic fragment.
  • In some embodiments, the heparanase isoform 1 polypeptide or immunogenic fragment, the heparanase isoform 2 polypeptide or immunogenic fragment and the SMAD4 polypeptide or immunogenic fragment are each 8-29 amino acids in length. In some embodiments, the heparanase isoform 1 polypeptide comprises the amino acid sequence of SEQ ID NO:1 and the heparanase isoform 2 polypeptide comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the SMAD4 polypeptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the one or more immunogenic fragments consist of about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the total number of amino acids of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In some embodiments, one or more immunogenic fragments consist of SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8 lacking about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids. In some embodiments, the heparanase isoform 1 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:6 and wherein the heparanase isoform 1 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:7. In some embodiments, the SMAD4 polypeptide comprises an amino acid sequence at least 85%, 90%, 95%, 97%, or 99% identical to SEQ ID NO:8. In some embodiments, the compositions further comprises an adjuvant.
  • In another aspect of the invention, methods of treating cancer comprises administering to a subject the immunogenic compositions described herein. In some embodiments, the subject has or is at risk of cancer, and/or exhibits one or more signs or symptoms of cancer, and/or exhibits one or more risk factors for cancer. In some embodiments, the cancer is colorectal cancer, melanoma, or lung cancer.
  • In another aspect of the invention, methods of inducing an immune response in a subject, comprise administering to a subject the immunogenic compositions described herein. In some embodiment, the immune response comprises activation of one or more lymphocytes. In some embodiments, the one or more lymphocytes comprise CD4+ T cells. In some embodiments, the one or more lymphocytes comprise CD8+ T cells. In some embodiments, the one or more lymphocytes comprise NKT cells, gamma-delta T cells, or NK cells. In some embodiments, the one or more lymphocytes comprise any combination of CD4+ T cells, CD8+ T cells, NKT cells, gamma-delta T cells, and NK cells. In some embodiments, the immune response comprises an increased expression and/or secretion of one or more immune mediators relative to a control. In some embodiments, the lymphocyte signaling molecule is selected from among immune mediators. In some embodiments, the one or more immune mediators are cytokines. In some embodiments, the one or more cytokines are selected from TRAIL, IFN-gamma, IL-12p70, IL-2, TNF-alpha, MIP1-alpha, MIP1-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3-alpha, MCP1, and fractalkine. In some embodiments, the one or more immune mediators are soluble mediators. In some embodiments, the one or more soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin. In some embodiments, the one or more immune mediators are cell surface markers. In some embodiments, the one or more cell surface markers are selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD1 (CD279), FoxP3, TIGIT, CD160, BTLA, 2B4 (CD244), and KLRG1. In some embodiments, a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher than a control level. In some embodiments, a level of one or more expressed or secreted immune mediators that is at least one, two, or three standard deviations higher than the mean of a control level indicates lymphocyte activation. In some embodiments, a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) higher or lower than a median response level to a control indicates lymphocyte activation. In some embodiments, the immune response comprises a humoral response and/or a cellular response. In some embodiments, humoral response comprises an increase in magnitude of response or fold rise from baseline of antigen specific immunoglobulin G (IgG) levels and/or of antigen specific neutralizing antibody levels. In some embodiments, the humoral response comprises a 4-fold or greater rise in IgG titer from baseline. In some embodiments, the humoral response comprises a 2-fold or greater rise in 50% neutralizing antibody titer from baseline. In some embodiments, the cellular response comprises secretion of granzyme B (GrB). In some embodiments, the cellular response comprises an increase in magnitude of response or fold rise from baseline of granzyme B (GrB) levels. In some embodiments, the cellular response comprises an increase in IFN-gamma secretion for T cells. In some embodiments, the subject has or is at risk of cancer, and/or exhibits one or more signs or symptoms of cancer, and/or exhibits one or more risk factors for cancer. In some embodiments, the cancer is colorectal cancer, melanoma, or lung cancer.
  • In another aspect, the disclosure features a method for manufacturing an immunogenic composition, the method comprising combining one or more antigens identified by any method described herein and a carrier.
  • In some embodiments, the antigen is produced using recombinant DNA technology in a suitable host cell. In some embodiments, the method comprises formulating the immunogenic composition as a pharmaceutical composition.
  • In another aspect, the disclosure features a method for manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: a. providing, preparing, or obtaining a plurality of antigenic compositions comprising a plurality of antigens, each composition comprising a different antigen; b. providing, preparing, or obtaining a target response profile, wherein the target response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; c. providing, preparing, or obtaining a subject response profile, wherein the subject response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; d. comparing the target response profile to the subject response profile; e. selecting one or more antigens based on the comparison; and f. formulating at least a portion of one or more antigenic compositions comprising the one or more selected antigens as a pharmaceutical composition.
  • In some embodiments, the selecting step comprises selecting one or more antigens that increase expression or secretion of immune mediators associated with a beneficial response to cancer, and/or one or more antigens that inhibit and/or suppress expression or secretion of immune mediators associated with deleterious or not beneficial responses to cancer. In some embodiments, the plurality of antigenic compositions are in solution, lyophilized, or on a synthetic matrix.
  • In another aspect, the disclosure features a method of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: preparing one or more antigens, or fragments thereof, identified by any of the method described herein; combining one or more antigens, or fragments thereof, wherein the one or more antigens or fragments thereof are selected according to the subject's response profile; and formulating the immunogenic composition as a pharmaceutical composition.
  • In another aspect, the disclosure features a method of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: preparing one or more antigens, or fragments thereof, identified by any method described herein; combining one or more antigens, or fragments thereof, wherein the one or more antigens or fragments thereof are selected according to whether or not the one or more antigens have been shown to stimulate, inhibit and/or suppress and/or have minimal effect on level of expression and/or secretion of one or more immune mediators by the subject's lymphocytes; and formulating the immunogenic composition as a pharmaceutical composition.
  • In another aspect, the disclosure features a method of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: preparing one or more antigens, or fragments thereof, identified by any method described herein;
  • combining one or more antigens, or fragments thereof, wherein the one or more antigens or fragments thereof are selected according to the subject's response profile; and formulating the immunogenic composition as a pharmaceutical composition.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.
  • FIG. 1 is a graph showing IFNγ secreted in supernatants by T cells from a representative melanoma patient who received immune checkpoint blockade therapy. The T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor-associated antigens.
  • FIG. 2 is a graph showing the number of T cell antigens that stimulated cytokine secretion in supernatants by CD4+ T cells from melanoma patients who were non-responders or responders to immune checkpoint blockade therapy. The T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor associated antigens.
  • FIG. 3 is a graph showing the number of T cell antigens that stimulated cytokine secretion in supernatants by CD8+ T cells from melanoma patients who were non-responders or responders to immune checkpoint blockade therapy. The T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor-associated antigens.
  • FIG. 4 is a scatter plot showing good alignment between replicate measurements for cytokines secreted by T cells from a representative NSCLC patient after stimulation by autologous antigen presenting cells pulsed with E. coli expressing putative neoantigens.
  • FIG. 5 is a graph showing results for IFNγ and TNFα secretion from CD8+ T cells from a representative NSCLC patient, collected pre- and post-checkpoint blockade therapy, after co-culture with autologous antigen presenting cells pulsed with E. coli expressing putative neoantigens.
  • FIG. 6 is a graph showing results for IFNγ and TNFα secretion from CD4+ T cells from a representative NSCLC patient, collected pre- and post-checkpoint blockade therapy, after co-culture with autologous antigen presenting cells pulsed with E. coli expressing putative neoantigens.
  • FIG. 7 is a Venn diagram showing limited overlap between CD8+-specific T cell neoantigens from a representative NSCLC patient, identified using methods of the disclosure and epitope prediction algorithms.
  • FIG. 8 is a schematic showing epitope predictions had a high false positive rate, missed relevant antigens and failed to identify suppressive and/or inhibitory neoantigens.
  • FIG. 9 is a graph showing IFNγ and TNF-α secreted in supernatants by T cells from a representative patient with colorectal cancer. The T cells were co-cultured with autologous antigen presenting cells pulsed with E. coli expressing various tumor-associated antigens. NG=neon green.
  • FIG. 10 is a graph showing the percentage of colorectal cancer patients who responded to each TAA, as measured by IFNγ secretion that exceeded three standard deviations of the mean negative control response.
  • FIG. 11 is a graph showing results for IFNγ and TNF-α secretion from CD8+ T cells from a patient with colorectal carcinoma after co-culture with antigen presenting cells pulsed with E. coli expressing 31 mutations unique to the patient.
  • FIGS. 12A and 12B are Venn diagrams representing the limited overlap between CD8+-specific T cell neoantigens identified by ATLAS and epitope prediction algorithms. FIG. 12A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ≤1 for the mutant peptide but not for wild-type. FIG. 12B represents epitopes predicted to have binding affinity below 500 nM or an IEDB percentile rank of ≤1, irrespective of the wild-type counterpart predictions.
  • FIGS. 13A and 13B are Venn diagrams representing the limited overlap between CD8+-specific T cell inhibitory and/or suppressive neoantigens identified by ATLAS and epitope prediction algorithms. FIG. 13A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ≤1 for the mutant peptide but not for wild-type. FIG. 13B represents epitopes predicted to have binding affinity below 500 nM or an IEDB percentile rank of ≤1, irrespective of the wild-type counterpart predictions.
  • FIG. 14 is a graph showing response profiles to 25 CRC-associated TAAs across CRC patients with all stages of disease using TNF-α and IFN-γ secretion as an indicator for a recall response to a putative antigen.
  • FIGS. 15A and 15B are graphs showing the high frequency of T cell stimulatory responses to three novel ATLAS-identified TAAs in comparison to three TAAs that are or were in clinical development as a therapeutic vaccine.
  • FIG. 16 is a graph showing stimulatory response rates to 4 selected TAAs for both CD4+ and CD8+ T cell subsets from CRC patients with early or late stage disease and TNF-α and IFN-γ cytokine release.
  • FIG. 17 is a graph showing normalized cytokine concentrations released in response to 4 selected TAAs in healthy individuals and donors with various disease states (polyps or CRC) for CD4+ and CD8+ T cell subsets and for TNF-α and IFN-γ release.
  • FIG. 18 is a graph showing an exemplary empirical determination of T cell responses to profiled TAAs. Exemplary data is shown for a single lung cancer patient. T cell responses are reported as natural log concentrations extrapolated from the MSD standard curve and normalized to the patient's response to a negative control protein.
  • FIG. 19 is a graph showing shows frequent CD4+ T cell responses to two novel TAAs compared to previously described TAAs (NY-ESO-1, MUC1, and MAGEA3). Each point represents a patient's response to that TAA, normalized to the patient's response to a negative control protein. Stimulatory responses are colored black.
  • FIG. 20 is a graph showing CD4+ and CD8+ T cell responses to a broad range of TAAs from lung cancer patients.
  • FIG. 21 is a graph showing inhibitory and/or suppressive T cell responses detected in most profiled TAAs across lung cancer patients.
  • FIG. 22 is a graph showing a neoantigen screen with ATLAS identifying patient-specific CD4+ and CD8+ T cell responses. Each dot represents a technical replicate. Horizontal dotted lines indicate the cutoffs used to define stimulatory neoantigens and inhibitory and/or suppressive neoantigens at +3 and −3 Median Absolute Deviations (MADs), respectively.
  • FIGS. 23A, 23B, 23C, and 23D show that algorithm prediction of MHC Class I binding does not accurately predict CD8+ T cell responses or types of response. FIGS. 23A and 23C show the total numbers and overlap of neoantigens predicted by algorithm and observed in ATLAS. FIGS. 23B and 23D show the break-down of predictions by strong binding (<150 nM), weak binding (<500 nM), or non-binding (>=500 nM). There is no enrichment of either stimulatory or inhibitory and/or suppressive responses in CD8+ T cells across binding prediction groups.
  • FIGS. 24A and 24B are graphs showing that CD8+ T cell responses identified by ATLAS to candidate neoantigens are not enriched for any mutation type.
  • FIGS. 25A and 25B are graphs showing DNA mutant allele frequency is not associated with CD8+ T cell response frequency.
  • FIGS. 26A and 26B are graphs showing detection of a mutation in RNA does not predict whether the candidate neoantigen elicits a recall response in CD8+ T cells.
  • FIGS. 27A and 27B are graphs showing that CD8+ T cell responses identified by ATLAS to candidate neoantigens do not correlate with gene expression.
  • FIG. 28 is a graph illustrating the different cytokine response profiles elicited by 6 representative neoantigens in a screen of CD8+ T cells from a single patient.
  • FIG. 29 is a graph showing CD8+ T cell data for healthy donors and cancer patients. When analyzed by IFNγ secretion, there was a large inhibitory response in the healthy donor cohort, that greatly exceeded the inhibitory responses in the cancer patient cohort. Conversely, there was a greater median inhibitory response in the cancer cohort when TNFα secretion was considered.
    •  DEFINITIONS
  • Activate: As used herein, a peptide presented by an antigen presenting cell (APC) “activates” a lymphocyte if lymphocyte activity is detectably modulated after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur. Any indicator of lymphocyte activity can be evaluated to determine whether a lymphocyte is activated, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers.
  • Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be systemic or local. In some embodiments, administration may be enteral or parenteral. In some embodiments, administration may be by injection (e.g., intramuscular, intravenous, or subcutaneous injection). In some embodiments, injection may involve bolus injection, drip, perfusion, or infusion. In some embodiments administration may be topical. Those skilled in the art will be aware of appropriate administration routes for use with particular therapies described herein, for example from among those listed on www.fda.gov, which include auricular (otic), buccal, conjunctival, cutaneous, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronal, intracorporus cavernosum, intradermal, intranodal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastic, intragingival, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (e.g., inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, ureteral, urethral, or vaginal. In some embodiments, administration may involve electro-osmosis, hemodialysis, infiltration, iontophoresis, irrigation, and/or occlusive dressing. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing.
  • Antigen: The term “antigen”, as used herein, refers to a molecule (e.g., a polypeptide) that elicits a specific immune response. Antigen-specific immunological responses, also known as adaptive immune responses, are mediated by lymphocytes (e.g., T cells, B cells, NK cells) that express antigen receptors (e.g., T cell receptors, B cell receptors). In certain embodiments, an antigen is a T cell antigen, and elicits a cellular immune response. In certain embodiments, an antigen is a B cell antigen, and elicits a humoral (i.e., antibody) response. In certain embodiments, an antigen is both a T cell antigen and a B cell antigen. As used herein, the term “antigen” encompasses both a full-length polypeptide as well as a portion or immunogenic fragment of the polypeptide, and a peptide epitope within the polypeptides (e.g., a peptide epitope bound by a Major Histocompatibility Complex (MHC) molecule (e.g., MHC class I, or MHC class II)).
  • Antigen presenting cell: An “antigen presenting cell” or “APC” refers to a cell that presents peptides on MHC class I and/or MHC class II molecules for recognition by T cells. APC include both professional APC (e.g., dendritic cells, macrophages, B cells), which have the ability to stimulate naïve lymphocytes, and non-professional APC (e.g., fibroblasts, epithelial cells, endothelial cells, glial cells). In certain embodiments, APC are able to internalize (e.g., endocytose) members of a library (e.g., cells of a library of bacterial cells) that express heterologous polypeptides as candidate antigens.
  • Autolysin polypeptide: An “autolysin polypeptide” is a polypeptide that facilitates or mediates autolysis of a cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell. In some embodiments, an autolysin polypeptide is a bacterial autolysin polypeptide. Autolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in GenBank® under Acc. Nos. NP 388823.1, NP 266427.1, and POAGC3.1.
  • Cancer: As used herein, the term “cancer” refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they display an abnormally elevated proliferation rate and/or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a cancer may be characterized by one or more tumors. Those skilled in the art are aware of a variety of types of cancer including, for example, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, myeloid leukemia), lymphoma (e.g., Burkitt lymphoma [non-Hodgkin lymphoma], cutaneous T cell lymphoma, Hodgkin lymphoma, mycosis fungoides, Sezary syndrome, AIDS-related lymphoma, follicular lymphoma, diffuse large B-cell lymphoma), melanoma, merkel cell carcinoma, mesothelioma, myeloma (e.g., multiple myeloma), myelodysplastic syndrome, papillomatosis, paraganglioma, pheochromacytoma, pleuropulmonary blastoma, retinoblastoma, sarcoma (e.g., Ewing sarcoma, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, uterine sarcoma, vascular sarcoma), Wilms' tumor, and/or cancer of the adrenal cortex, anus, appendix, bile duct, bladder, bone, brain, breast, bronchus, central nervous system, cervix, colon, endometrium, esophagus, eye, fallopian tube, gall bladder, gastrointestinal tract, germ cell, head and neck, heart, intestine, kidney (e.g., Wilms' tumor), larynx, liver, lung (e.g., non-small cell lung cancer, small cell lung cancer), mouth, nasal cavity, oral cavity, ovary, pancreas, rectum, skin, stomach, testes, throat, thyroid, penis, pharynx, peritoneum, pituitary, prostate, rectum, salivary gland, ureter, urethra, uterus, vagina, or vulva.
  • Cytolysin polypeptide: A “cytolysin polypeptide” is a polypeptide that has the ability to form pores in a membrane of a eukaryotic cell. A cytolysin polypeptide, when expressed in host cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell, facilitates release of host cell components (e.g., host cell macromolecules, such as host cell polypeptides) into the cytosol of the internalizing cell. In some embodiments, a cytolysin polypeptide is bacterial cytolysin polypeptide. In some embodiments, a cytolysin polypeptide is a cytoplasmic cytolysin polypeptide. Cytolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in U.S. Pat. No. 6,004,815, and in GenBank® under Acc. Nos. NP_463733.1, NP_979614, NP_834769, YP_084586, YP_895748, YP_694620, YP_012823, NP_346351, YP_597752, BAB41212.2, NP_561079.1, YP_001198769, and NP_359331.1.
  • Cytoplasmic cytolysin polypeptide: A “cytoplasmic cytolysin polypeptide” is a cytolysin polypeptide that has the ability to form pores in a membrane of a eukaryotic cell, and that is expressed as a cytoplasmic polypeptide in a bacterial cell. A cytoplasmic cytolysin polypeptide is not significantly secreted by a bacterial cell. Cytoplasmic cytolysin polypeptides can be provided by a variety of means. In some embodiments, a cytoplasmic cytolysin polypeptide is provided as a nucleic acid encoding the cytoplasmic ccytolysin polypeptide. In some embodiments, a cytoplasmic cytolysin polypeptide is provided attached to a bead. In some embodiments, a cytoplasmic cytolysin polypeptide has a sequence that is altered relative to the sequence of a secreted cytolysin polypeptide (e.g., altered by deletion or alteration of a signal sequence to render it nonfunctional). In some embodiments, a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a secretion-incompetent cell. In some embodiments, a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a cell that does not recognize and mediate secretion of a signal sequence linked to the cytolysin polypeptide. In some embodiments, a cytoplasmic cytolysin polypeptide is a bacterial cytolysin polypeptide.
  • Heterologous: The term “heterologous”, as used herein to refer to genes or polypeptides, refers to a gene or polypeptide that does not naturally occur in the organism in which it is present and/or being expressed, and/or that has been introduced into the organism by the hand of man. In some embodiments, a heterologous polypeptide is a tumor antigen described herein.
  • Immune mediator: As used herein, the term “immune mediator” refers to any molecule that affects the cells and processes involved in immune responses. Immune mediators include cytokines, chemokines, soluble proteins, and cell surface markers.
  • Improve, increase, inhibit, stimulate, suppress, or reduce: As used herein, the terms “improve”, “increase”, “inhibit”, “stimulate”, “suppress”, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. The effect of a particular agent or treatment may be direct or indirect. In some embodiments, an appropriate reference measurement may be or may comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. In some embodiments, a peptide presented by an antigen presenting cell (APC) “stimulates” or is “stimulatory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control. In some embodiments, a peptide presented by an antigen presenting cell “suppresses”, “inhibits” or is “inhibitory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with deleterious or non-beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control.
  • Invasin polypeptide: An “invasin polypeptide” is a polypeptide that facilitates or mediates uptake of a cell (e.g., a bacterial cell) by a eukaryotic cell. Expression of an invasin polypeptide in a noninvasive bacterial cell confers on the cell the ability to enter a eukaryotic cell. In some embodiments, an invasin polypeptide is a bacterial invasin polypeptide. In some embodiments, an invasin polypeptide is a Yersinia invasin polypeptide (e.g., a Yersinia invasin polypeptide comprising a sequence disclosed in GenBank® under Acc. No. YP_070195.1).
  • Listeriolysin O (LLO): The terms “listeriolysin O” or “LLO” refer to a listeriolysin O polypeptide of Listeria monocytogenes and truncated forms thereof that retain pore-forming ability (e.g., cytoplasmic forms of LLO, including truncated forms lacking a signal sequence). In some embodiments, an LLO is a cytoplasmic LLO. Exemplary LLO sequences are shown in Table 1, below.
  • Polypeptide: The term “polypeptide”, as used herein, generally has its art-recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate, however, that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (i.e., fragments retaining at least one activity) and immunogenic fragments of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Other regions of similarity and/or identity can be determined by those of ordinary skill in the art by analysis of the sequences of various polypeptides.
  • Primary cells: As used herein, “primary cells” refers to cells from an organism that have not been immortalized in vitro. In some embodiments, primary cells are cells taken directly from a subject (e.g., a human). In some embodiments, primary cells are progeny of cells taken from a subject (e.g., cells that have been passaged in vitro). Primary cells include cells that have been stimulated to proliferate in culture.
  • Response: As used herein, in the context of a subject (a patient or experimental organism), “response”, “responsive”, or “responsiveness” refers to an alteration in a subject's condition that occurs as a result of, or correlates with, treatment. In certain embodiments, a response is a beneficial response. In certain embodiments, a beneficial response can include stabilization of a subject's condition (e.g., prevention or delay of deterioration expected or typically observed to occur absent the treatment), amelioration (e.g., reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or improvement in the prospects for cure of the condition, etc. In certain embodiments, for a subject who has cancer, a beneficial response can include: the subject has a positive clinical response to cancer therapy or a combination of therapies; the subject has a spontaneous response to a cancer; the subject is in partial or complete remission from cancer; the subject has cleared a cancer; the subject has not had a relapse, recurrence or metastasis of a cancer; the subject has a positive cancer prognosis; the subject has not experienced toxic responses or side effects to a cancer therapy or combination of therapies. In certain embodiments, for a subject who had cancer, the beneficial responses occurred in the past, or are ongoing.
  • In certain embodiments, a response is a deleterious or non-beneficial response. In certain embodiments, a deleterious or non-beneficial response can include deterioration of a subject's condition, lack of amelioration (e.g., no reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or degradation in the prospects for cure of the condition, etc. In certain embodiments, for a subject who has cancer, a deleterious or non-beneficial response can include: the subject has a negative clinical response to cancer therapy or a combination of therapies; the subject is not in remission from cancer; the subject has not cleared a cancer; the subject has had a relapse, recurrence or metastasis of a cancer; the subject has a negative cancer prognosis; the subject has experienced toxic responses or side effects to a cancer therapy or combination of therapies. In certain embodiments, for a subject who had cancer, the deleterious or non-beneficial responses occurred in the past, or are ongoing.
  • As used herein, in the context of a cell, organ, tissue, or cell component, e.g., a lymphocyte, “response”, “responsive”, or “responsiveness” refers to an alteration in cellular activity that occurs as a result of, or correlates with, administration of or exposure to an agent, e.g. a tumor antigen. In certain embodiments, a beneficial response can include increased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject. In certain embodiments, a beneficial response can include decreased expression and/or secretion of immune mediators associated with negative clinical response or outcomes in a subject. In certain embodiments, a deleterious or non-beneficial response can include increased expression and/or secretion of immune mediators associated with negative clinical responses or outcomes in a subject. In certain embodiments, a deleterious or non-beneficial response can include decreased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject. In certain embodiments, a response is a clinical response. In certain embodiments, a response is a cellular response. In certain embodiments, a response is a direct response. In certain embodiments, a response is an indirect response. In certain embodiments, “non-response”, “non-responsive”, or “non-responsiveness” mean minimal response or no detectable response. In certain embodiments, a “minimal response” includes no detectable response. In certain embodiments, presence, extent, and/or nature of response can be measured and/or characterized according to particular criteria. In certain embodiments, such criteria can include clinical criteria and/or objective criteria. In certain embodiments, techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MM, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology. Where a response of interest is a response of a tumor to a therapy, ones skilled in the art will be aware of a variety of established techniques for assessing such response, including, for example, for determining tumor burden, tumor size, tumor stage, etc. Methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:e143-52. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of tumors, patients or experimental organism, and/or cells, organs, tissues, or cell components, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria.
  • Tumor: As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. In some embodiments, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments, a tumor may be a disperse tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.
    • DETAILED DESCRIPTION
  • Recent advances in immune checkpoint inhibitor therapies such as ipilimumab, nivolumab, and pembrolizumab for cancer immunotherapy have resulted in dramatic efficacy in subjects suffering from NSCLC, among other indications. Nivolumab and pembroluzimab have been approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for use in patients with advanced NSCLC who have previously been treated with chemotherapy. They have solidified the importance of T cell responses in control of tumors. Neoantigens, potential cancer rejection antigens that are entirely absent from the normal human genome, are postulated to be relevant to tumor control; however, attempts to define them and their role in tumor clearance has been hindered by the paucity of available tools to define them in a biologically relevant and unbiased way (Schumacher and Schreiber, 2015 Science 348:69-74, Gilchuk et al., 2015 Curr Opin Immunol 34:43-51)
  • Taking non-small cell lung carcinoma (NSCLC) as an example, whole exome sequencing of NSCLC tumors from patients treated with pembrolizumab showed that higher non-synonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival (Rizvi et al., (2015) Science 348(6230): 124-8). In this study, the median non-synonymous mutational burden of the discovery cohort was 209 and of the validation cohort was 200. However, simply because a mutation was identified by sequencing, does not mean that the epitope it creates can be recognized by a T cell or serves as a protective antigen for T cell responses (Gilchuk et al., 2015 Curr Opin Immunol 34:43-51), making the use of the word neoantigen somewhat of a misnomer. With 200 or more potential targets of T cells in NSCLC, it is not feasible to test every predicted epitope to determine which of the mutations serve as neoantigens, and which neoantigens are associated with clinical evidence of tumor control. Recently, a study by McGranahan et al., showed that clonal neoantigen burden and overall survival in primary lung adenocarcinomas are related. However, even enriching for clonal neoantigens results in potential antigen targets ranging from 50 to approximately 400 (McGranahan et al., 2016 Science 351:1463-69). Similar findings have been described for melanoma patients who have responded to ipilimumab therapy (Snyder et al., 2015 NEJM; Van Allen et al., 2015 Science) and in patients with mismatch-repair deficient colorectal cancer who were treated with pembrolizumab (Le et al., 2015 NEJM).
  • The present disclosure provides methods and systems for the rapid identification of tumor antigens (e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs)) that elicit T cell responses and particularly that elicit human T cell responses, as well as polypeptides that are potential tumor antigens. For purposes of this disclosure, “tumor antigens” includes both tumor antigens and potential tumor antigens. As described herein, methods of the present disclosure identified stimulatory tumor antigens that were not identified by known algorithms. Further, methods of the present disclosure identified suppressive and/or inhibitory tumor antigens that are not identifiable by known algorithms. Methods of the present disclosure also identified polypeptides that are potential tumor antigens, i.e., polypeptides that activate T cells of non-cancerous subjects, but not T cells of subjects suffering from cancer. The present disclosure also provides methods of selecting tumor antigens and potential tumor antigens, methods of using the selected tumor antigens and potential tumor antigens, immunogenic compositions comprising the selected tumor antigens and potential tumor antigens, and methods of manufacturing immunogenic compositions. The present disclosure also provides methods of evaluating an immune response in a cancer subject, e.g., for identifying or selecting subjects for initiation, continuation, modification, and/or discontinuation of cancer therapy.
    • Library Generation
  • A library is a collection of members (e.g., cells or non-cellular particles, such as virus particles, liposomes, or beads (e.g., beads coated with polypeptides, such as in vitro translated polypeptides, e.g., affinity beads, e.g., antibody coated beads, or NTA-Ni beads bound to polypeptides of interest). According to the present disclosure, members of a library include (e.g., internally express or carry) polypeptides of interest described herein. In some embodiments, members of a library are cells that internally express polypeptides of interest described herein. In some embodiments, members of a library which are particles carry, and/or are bound to, polypeptides of interest. Use of a library in an assay system allows simultaneous evaluation in vitro of cellular responses to multiple candidate antigens. According to the present disclosure, a library is designed to be internalized by human antigen presenting cells so that peptides from library members, including peptides from internally expressed polypeptides of interest, are presented on MEW molecules of the antigen presenting cells for recognition by T cells.
  • Libraries can be used in assays that detect peptides presented by human MHC class I and MHC class II molecules. Polypeptides expressed by the internalized library members are digested in intracellular endocytic compartments (e.g., phagosomes, endosomes, lysosomes) of the human cells and presented on MHC class II molecules, which are recognized by human CD4+ T cells. In some embodiments, library members include a cytolysin polypeptide, in addition to a polypeptide of interest. In some embodiments, library members include an invasin polypeptide, in addition to the polypeptide of interest. In some embodiments, library members include an autolysin polypeptide, in addition to the polypeptide of interest. In some embodiments, library members are provided with cells that express a cytolysin polypeptide (i.e., the cytolysin and polypeptide of interest are not expressed in the same cell, and an antigen presenting cell is exposed to members that include the cytolysin and members that include the polypeptide of interest, such that the antigen presenting cell internalizes both, and such that the cytolysin facilitates delivery of polypeptides of interest to the MHC class I pathway of the antigen presenting cell). A cytolysin polypeptide can be constitutively expressed in a cell, or it can be under the control of an inducible expression system (e.g., an inducible promoter). In some embodiments, a cytolysin is expressed under the control of an inducible promoter to minimize cytotoxicity to the cell that expresses the cytolysin.
  • Once internalized by a human cell, a cytolysin polypeptide perforates intracellular compartments in the human cell, allowing polypeptides expressed by the library members to gain access to the cytosol of the human cell. Polypeptides released into the cytosol are presented on MHC class I molecules, which are recognized by CD8+ T cells.
  • A library can include any type of cell or particle that can be internalized by and deliver a polypeptide of interest (and a cytolysin polypeptide, in applications where a cytolysin polypeptide is desirable) to, antigen presenting cells for use in methods described herein. Although the term “cell” is used throughout the present specification to refer to a library member, it is understood that, in some embodiments, the library member is a non-cellular particle, such as a virus particle, liposome, or bead. In some embodiments, members of the library include polynucleotides that encode the polypeptide of interest (and cytolysin polypeptide), and can be induced to express the polypeptide of interest (and cytolysin polypeptide) prior to, and/or during internalization by antigen presenting cells.
  • In some embodiments, the cytolysin polypeptide is heterologous to the library cell in which it is expressed, and facilitates delivery of polypeptides expressed by the library cell into the cytosol of a human cell that has internalized the library cell. Cytolysin polypeptides include bacterial cytolysin polypeptides, such as listeriolysin O (LLO), streptolysin O (SLO), and perfringolysin O (PFO). Additional cytolysin polypeptides are described in U.S. Pat. No. 6,004,815. In certain embodiments, library members express LLO. In some embodiments, a cytolysin polypeptide is not significantly secreted by the library cell (e.g., less than 20%, 10%, 5%, or 1% of the cytolysin polypeptide produced by the cell is secreted). For example, the cytolysin polypeptide is a cytoplasmic cytolysin polypeptide, such as a cytoplasmic LLO polypeptide (e.g., a form of LLO which lacks the N-terminal signal sequence, as described in Higgins et al., Mol. Microbiol. 31(6):1631-1641,1999). Exemplary cytolysin polypeptide sequences are shown in Table 1. The listeriolysin O (43-25) sequence shown in the second row of Table 1 has a deletion of residues 3-25, relative to the LLO sequence in shown in the first row of Table 1, and is a cytoplasmic LLO polypeptide. In some embodiments, a cytolysin is expressed constitutively in a library host cell. In other embodiments, a cytolysin is expressed under the control of an inducible promoter. Cytolysin polypeptides can be expressed from the same vector, or from a different vector, as the polypeptide of interest in a library cell.
  • TABLE 1
    Exemplary Cytolysin Polypeptides
    Polypeptide
    Polypeptide Name Accession No.
    (species) GI No. Polypeptide Sequence
    listeriolysin O NP_463733.1 MKKIMLVFITLILVSLPIAQQTEAKDASAFNKENSISSMAPPASP
    (Listeria GI: 16802248 PASPKTPIEKKHADEIDKYIQGLDYNKNNVLVYHGDAVTNVPPRK
    monocytogenes) GYKDGNEYIVVEKKKKSINQNNADIQVVNAISSLTYPGALVKANS
    ELVENQPDVLPVKRDSLILSIDLPGMTNQDNKIVVKNATKSNVNN
    AVNTLVERWNEKYAQAYPNVSAKIDYDDEMAYSESQLIAKFGTAF
    KAVNNSLNVNFGAISEGKMQEEVISFKQIYYNVNVNEPTRPSRFF
    GKAVTKEQLQALGVNAENPPAYISSVAYGRQVYLKLSTNSHSTKV
    KAAFDAAVSGKSVSGDVELTNIIKNSSFKAVIYGGSAKDEVQIID
    GNLGDLRDILKKGATFNRETPGVPIAYTTNFLKDNELAVIKNNSE
    YIETTSKAYTDGKINIDHSGGYVAQFNISWDEVNYDPEGNEIVQH
    KNWSENNKSKLAHFTSSIYLPGNARNINVYAKECTGLAWEWWRTV
    IDDRNLPLVKNRNISIWGTTLYPKYSNKVDNPIE (SEQ ID
    NO: 1)
    listeriolysin O MKDASAFNKENSISSMAPPASPPASPKTPIEKKHADEIDKYIQGL
    (Δ3-25) DYNKNNVLVYHGDAVTNVPPRKGYKDGNEYIVVEKKKKSINQNNA
    DIQVVNAISSLTYPGALVKANSELVENQPDVLPVKRDSLTLSIDL
    PGMTNQDNKIVVKNATKSNVNNAVNTLVERWNEKYAQAYPNVSAK
    IDYDDEMAYSESQLIAKEGTAFKAVNNSLNVNFGAISEGKMQEEV
    ISFKQIYYNVNVNEPTRPSRFFGKAVTKEQLQALGVNAENPPAYI
    SSVAYGRQVYLKLSTNSHSTKVKAAFDAAVSGKSVSGDVELTNII
    KNSSFKAVIYGGSAKDEVQIIDGNLGDLRDILKKGATFNRETPGV
    PIAYTTNFLKDNELAVIKNNSEYIETTSKAYTDGKINIDHSGGYV
    AQFNISWDEVNYDPEGNEIVQHKNWSENNKSKLAHFTSSIYLPGN
    ARNINVYAKECTGLAWEWWRTVIDDRNLPLVKNRNISIWGTTLYP
    KYSNKVDNPIE(SEQ ID NO: 2)
    streptolysin O BAB41212.2 MSNKKTFKKYSRVAGLLTAALIIGNLVTANAESNKQNTASTETTT
    (Streptococcus GI: 71061060 TSEQPKPESSELTIEKAGQKMDDMLNSNDMIKLAPKEMPLESAEK
    pyogenes) EEKKSEDKKKSEEDHTEEINDKIYSLNYNELEVLAKNGETIENFV
    PKEGVKKADKFIVIERKKKNINTTPVDISIIDSVTDRTYPAALQL
    ANKGFTENKPDAVVTKRNPQKIHIDLPGMGDKATVEVNDPTYANV
    STAIDNLVNQWHDNYSGGNTLPARTQYTESMVYSKSQIEAALNVN
    SKILDGTLGIDFKSISKGEKKVMIAAYKQIFYTVSANLPNNPADV
    FDKSVTFKDLQRKGVSNEAPPLFVSNVAYGRTVFVKLETSSKSND
    VEAAFSAALKGTDVKTNGKYSDILENSSFTAVVLGGDAAEHNKVV
    TKDFDVIRNVIKDNATFSRKNPAYPISYTSVFLKNNKIAGVNNRT
    EYVETTSTEYTSGKINLSHQGAYVAQYEILWDEINYDDKGKEVIT
    KRRWDNNWYSKTSPFSTVIPLGANSRNIRIMARECTGLAWEWWRK
    VIDERDVKLSKEINVNISGSTLSPYGSITYK (SEQ ID NO: 3)
    perfringolysin O NP_561079.1 MIRFKKTKLIASIAMALCLFSQPVISFSKDITDKNQSIDSGISSL
    (Clostridium GI: 18309145 SYNRNEVLASNGDKIESFVPKEGKKTGNKFIVVERQKRSLTTSPV
    perfringens) DISIIDSVNDRTYPGALQLADKAFVENRPTILMVKRKPININIDL
    PGLKGENSIKVDDPTYGKVSGAIDELVSKWNEKYSSTHTLPARTQ
    YSESMVYSKSQISSALNVNAKVLENSLGVDFNAVANNEKKVMILA
    YKQIFYTVSADLPKNPSDLFDDSVTFNDLKQKGVSNEAPPLMVSN
    VAYGRTIYVKLETTSSSKDVQAAFKALIKNTDIKNSQQYKDIYEN
    SSFTAVVLGGDAQEHNKVVTKDFDEIRKVIKDNATFSTKNPAYPI
    SYTSVFLKDNSVAAVHNKTDYIETTSTEYSKGKINLDHSGAYVAQ
    FEVAWDEVSYDKEGNEVLTHKTWDGNYQDKTAHYSTVIPLEANAR
    NIRIKARECTGLAWEWWRDVISEYDVPLTNNINVSIWGTTLYPGS
    SITYN (SEQ ID NO: 4)
    Pneumolysin NP_359331.1 MANKAVNDFILAMNYDKKKLLTHQGESIENRFIKEGNQLPDEFVV
    (Streptococcus GI: 933687 IERKKRSLSTNTSDISVTATNDSRLYPGALLVVDETLLENNPTLL
    pneumoniae) AVDRAPMTYSIDLPGLASSDSFLQVEDPSNSSVRGAVNDLLAKWH
    QDYGQVNNVPARMQYEKITAHSMEQLKVKFGSDFEKTGNSLDIDF
    NSVHSGEKQIQIVNFKQIYYTVSVDAVKNPGDVFQDTVTVEDLKQ
    RGISAERPLVYISSVAYGRQVYLKLETTSKSDEVEAAFEALIKGV
    KVAPQTEWKQILDNTEVKAVILGGDPSSGARVVTGKVDMVEDLIQ
    EGSRFTADHPGLPISYTTSFLRDNVVATFQNSTDYVETKVTAYRN
    GDLLLDHSGAYVAQYYITWDELSYDHQGKEVLTPKAWDRNGQDLT
    AHFTTSIPLKGNVRNLSVKIRECTGLAWEWWRTVYEKTDLPLVRK
    RTISIWGTTLYPQVEDKVEND (SEQ ID NO: 5)
  • In some embodiments, a library member (e.g., a library member which is a bacterial cell) includes an invasin that facilitates uptake by the antigen presenting cell. In some embodiments, a library member includes an autolysin that facilitates autolysis of the library member within the antigen presenting cell. In some embodiments, a library member includes both an invasin and an autolysin. In some embodiments, a library member which is an E. coli cell includes an invasin and/or an autolysin. In various embodiments, library cells that express an invasin and/or autolysin are used in methods that also employ non-professional antigen presenting cells or antigen presenting cells that are from cell lines. Isberg et al. (Cell, 1987, 50:769-778), Sizemore et al. (Science, 1995, 270:299-302) and Courvalin et al. (C.R. Acad. Sci. Paris, 1995, 318:1207-12) describe expression of an invasin to effect endocytosis of bacteria by target cells. Autolysins are described by Cao et al., Infect. Immun. 1998, 66(6): 2984-2986; Margot et al., J. Bacteriol. 1998, 180(3):749-752; Buist et al., Appl. Environ. Microbiol., 1997, 63(7):2722-2728; Yamanaka et al., FEMS Microbiol. Lett., 1997, 150(2): 269-275; Romero et al., FEMS Microbiol. Lett., 1993, 108(1):87-92; Betzner and Keck, Mol. Gen. Genet., 1989, 219(3): 489-491; Lubitz et al., I Bacteriol., 1984, 159(1):385-387; and Tomasz et al., I Bacteriol., 1988, 170(12): 5931-5934. In some embodiments, an autolysin has a feature that permits delayed lysis, e.g., the autolysin is temperature-sensitive or time-sensitive (see, e.g., Chang et al., 1995, 1 Bact. 177, 3283-3294; Raab et al., 1985, J Mol. Biol. 19, 95-105; Gerds et al., 1995, Mol. Microbiol. 17, 205-210). Useful cytolysins also include addiction (poison/antidote) autolysins, (see, e.g., Magnuson R, et al., 1996, J Biol. Chem. 271(31), 18705-18710; Smith A S, et al., 1997, Mol. Microbiol. 26(5), 961-970).
  • In some embodiments, members of the library include bacterial cells. In certain embodiments, the library includes non-pathogenic, non-virulent bacterial cells. Examples of bacteria for use as library members include E. coli, mycobacteria, Listeria monocytogenes, Shigella flexneri, Bacillus subtilis, or Salmonella.
  • In some embodiments, members of the library include eukaryotic cells (e.g., yeast cells). In some embodiments, members of the library include viruses (e.g., bacteriophages). In some embodiments, members of the library include liposomes. Methods for preparing liposomes that include a cytolysin and other agents are described in Kyung-Dall et al., U.S. Pat. No. 5,643,599. In some embodiments, members of the library include beads. Methods for preparing libraries comprised of beads are described, e.g., in Lam et al., Nature 354: 82-84, 1991, U.S. Pat. Nos. 5,510,240 and 7,262,269, and references cited therein.
  • In certain embodiments, a library is constructed by cloning polynucleotides encoding polypeptides of interest, or portions thereof, into vectors that express the polypeptides of interest in cells of the library. The polynucleotides can be synthetically synthesized. The polynucleotides can be cloned by designing primers that amplify the polynucleotides. Primers can be designed using available software, such as Primer3Plus (available the following URL: bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; see Rozen and Skaletsky, In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, N.J., pp. 365-386, 2000). Other methods for designing primers are known to those of skill in the art. In some embodiments, primers are constructed so as to produce polypeptides that are truncated, and/or lack hydrophobic regions (e.g., signal sequences or transmembrane regions) to promote efficient expression. The location of predicted signal sequences and predicted signal sequence cleavage sites in a given open reading frame (ORF) sequence can be determined using available software, see, e.g., Dyrløv et al., J. Mol. Biol., 340:783-795, 2004, and the following URL: cbs.dtu.dk/services/SignalP/). For example, if a signal sequence is predicted to occur at the N-terminal 20 amino acids of a given polypeptide sequence, a primer is designed to anneal to a coding sequence downstream of the nucleotides encoding the N-terminal 20 amino acids, such that the amplified sequence encodes a product lacking this signal sequence.
  • Primers can also be designed to include sequences that facilitate subsequent cloning steps. ORFs can be amplified directly from genomic DNA (e.g., genomic DNA of a tumor cell), or from polynucleotides produced by reverse transcription (RT-PCR) of mRNAs expressed by the tumor cell. RT-PCR of mRNA is useful, e.g., when the genomic sequence of interest contains intronic regions. PCR-amplified ORFs are cloned into an appropriate vector, and size, sequence, and expression of ORFs can be verified prior to use in immunological assays.
  • In some embodiments, a polynucleotide encoding a polypeptide of interest is linked to a sequence encoding a tag (e.g., an N-terminal or C-terminal epitope tag) or a reporter protein (e.g., a fluorescent protein). Epitope tags and reporter proteins facilitate purification of expressed polypeptides, and can allow one to verify that a given polypeptide is properly expressed in a library host cell, e.g., prior to using the cell in a screen. Useful epitope tags include, for example, a polyhistidine (His) tag, a V5 epitope tag from the P and V protein of paramyxovirus, a hemagglutinin (HA) tag, a myc tag, and others. In some embodiments, a polynucleotide encoding a polypeptide of interest is fused to a sequence encoding a tag which is a known antigenic epitope (e.g., an MHC class I- and/or MHC class II-restricted T cell epitope of a model antigen such as an ovalbumin), and which can be used to verify that a polypeptide of interest is expressed and that the polypeptide-tag fusion protein is processed and presented in antigen presentation assays. In some embodiments a tag includes a T cell epitope of a murine T cell (e.g., a murine T cell line). In some embodiments, a polynucleotide encoding a polypeptide of interest is linked to a tag that facilitates purification and a tag that is a known antigenic epitope. Useful reporter proteins include naturally occurring fluorescent proteins and their derivatives, for example, Green Fluorescent Protein (Aequorea Victoria) and Neon Green (Branchiostoma lanceolatum). Panels of synthetically derived fluorescent and chromogenic proteins are also available from commercial sources.
  • Polynucleotides encoding a polypeptide of interest are cloned into an expression vector for introduction into library host cells. Various vector systems are available to facilitate cloning and manipulation of polynucleotides, such as the Gateway® Cloning system (Invitrogen). As is known to those of skill in the art, expression vectors include elements that drive production of polypeptides of interest encoded by a polynucleotide in library host cells (e.g., promoter and other regulatory elements). In some embodiments, polypeptide expression is controlled by an inducible element (e.g., an inducible promoter, e.g., an IPTG- or arabinose-inducible promoter, or an IPTG-inducible phage T7 RNA polymerase system, a lactose (lac) promoter, a tryptophan (trp) promoter, a tac promoter, a trc promoter, a phage lambda promoter, an alkaline phosphatase (phoA) promoter, to give just a few examples; see Cantrell, Meth. in Mol. Biol., 235:257-276, Humana Press, Casali and Preston, Eds.). In some embodiments, polypeptides are expressed as cytoplasmic polypeptides. In some embodiments, the vector used for polypeptide expression is a vector that has a high copy number in a library host cell. In some embodiments, the vector used for expression has a copy number that is more than 25, 50, 75, 100, 150, 200, or 250 copies per cell. In some embodiments, the vector used for expression has a ColE1 origin of replication. Useful vectors for polypeptide expression in bacteria include pET vectors (Novagen), Gateway® pDEST vectors (Invitrogen), pGEX vectors (Amersham Biosciences), pPRO vectors (BD Biosciences), pBAD vectors (Invitrogen), pLEX vectors (Invitrogen), pMAL™ vectors (New England BioLabs), pGEMEX vectors (Promega), and pQE vectors (Qiagen). Vector systems for producing phage libraries are known and include Novagen T7Select® vectors, and New England Biolabs Ph.D.™ Peptide Display Cloning System.
  • In some embodiments, library host cells express (either constitutively, or when induced, depending on the selected expression system) a polypeptide of interest to at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total cellular protein. In some embodiments, the level a polypeptide available in or on a library member (e.g., cell, virus particle, liposome, bead) is such that antigen presenting cells exposed to a sufficient quantity of the library members are presented on MHC molecules polypeptide epitopes at a density that is comparable to the density presented by antigen presenting cells pulsed with purified peptides.
  • Methods for efficient, large-scale production of libraries are available. For example, site-specific recombinases or rare-cutting restriction enzymes can be used to transfer polynucleotides between expression vectors in the proper orientation and reading frame (Walhout et al., Meth. Enzymol. 328:575-592, 2000; Marsischky et al., Genome Res. 14:2020-202, 2004; Blommel et al., Protein Expr. Purif. 47:562-570, 2006).
  • For production of liposome libraries, expressed polypeptides (e.g., purified or partially purified polypeptides) can be entrapped in liposomal membranes, e.g., as described in Wassef et al., U.S. Pat. No. 4,863,874; Wheatley et al., U.S. Pat. No. 4,921,757; Huang et al., U.S. Pat. No. 4,925,661; or Martin et al., U.S. Pat. No. 5,225,212.
  • A library can be designed to include full length polypeptides and/or portions of polypeptides. Expression of full length polypeptides maximizes epitopes available for presentation by a human antigen presenting cell, thereby increasing the likelihood of identifying an antigen. However, in some embodiments, it is useful to express portions of polypeptides, or polypeptides that are otherwise altered, to achieve efficient expression. For example, in some embodiments, polynucleotides encoding polypeptides that are large (e.g., greater than 1,000 amino acids), that have extended hydrophobic regions, signal peptides, transmembrane domains, or domains that cause cellular toxicity, are modified (e.g., by C-terminal truncation, N-terminal truncation, or internal deletion) to reduce cytotoxicity and permit efficient expression a library cell, which in turn facilitates presentation of the encoded polypeptides on human cells. Other types of modifications, such as point mutations or codon optimization, may also be used to enhance expression.
  • The number of polypeptides included in a library can be varied. For example, in some embodiments, a library can be designed to express polypeptides from at least 5%, 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of ORFs in a target cell (e.g., tumor cell). In some embodiments, a library expresses at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2500, 5000, 10,000, or more different polypeptides of interest, each of which may represent a polypeptide encoded by a single full length polynucleotide or portion thereof.
  • In some embodiments, assays may focus on identifying antigens that are secreted polypeptides, cell surface-expressed polypeptides, or virulence determinants, e.g., to identify antigens that are likely to be targets of both humoral and cell mediated immune responses.
  • In addition to polypeptides of interest, libraries can include tags or reporter proteins that allow one to easily purify, analyze, or evaluate MHC presentation, of the polypeptide of interest. In some embodiments, polypeptides expressed by a library include C-terminal tags that include both an MHC class I and an MHC class II-restricted T cell epitope from a model antigen, such as chicken ovalbumin (OVA). Library protein expression and MHC presentation is validated using these epitopes. In some embodiments, the epitopes are OVA247-265 and OVA258-265 respectfully, corresponding to positions in the amino acid sequence found in GenBank® under Acc. No. NP_990483. Expression and presentation of linked ORFs can be verified with antigen presentation assays using T cell hybridomas (e.g., B3Z T hybridoma cells, which are H2-Kb restricted, and KZO T hybridoma cells, which are H2-Ak restricted) that specifically recognize these epitopes.
  • Sets of library members (e.g., bacterial cells) can be provided on an array (e.g., on a solid support, such as a 96-well plate) and separated such that members in each location express a different polypeptide of interest, or a different set of polypeptides of interest.
  • Methods of using library members for identifying T cell antigens are described in detail below. In addition to these methods, library members also have utility in assays to identify B cell antigens. For example, lysate prepared from library members that include polypeptides of interest can be used to screen a sample comprising antibodies (e.g., a serum sample) from a subject (e.g., a subject who has been exposed to an infectious agent of interest, a subject who has cancer, and/or a control subject), to determine whether antibodies present in the subject react with the polypeptide of interest. Suitable methods for evaluating antibody reactivity are known and include, e.g., ELISA assays.
    • Polypeptides of Interest
  • In some embodiments, methods and compositions described herein can be used to identify and/or detect immune responses to a polypeptide of interest. In some embodiments, a polypeptide of interest is encoded by an ORF from a target tumor cell, and members of a library include (e.g., internally express or carry) ORFs from a target tumor cell. In some such embodiments, a library can be used in methods described herein to assess immune responses to one or more polypeptides of interest encoded by one or more ORFs. In some embodiments, methods of the disclosure identify one or more polypeptides of interest as stimulatory antigens (e.g., that stimulate an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators). In some embodiments, methods of the disclosure identify one or more polypeptides of interest as antigens or potential antigens that have minimal or no effect on an immune response (e.g., expression and/or secretion of one or more immune mediators). In some embodiments, methods of the disclosure identify one or more polypeptides of interest as inhibitory and/or suppressive antigens (e.g., that inhibit, suppress, down-regulate, impair, and/or prevent an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators). In some embodiments, methods of the disclosure identify one or more polypeptides of interest as tumor antigens or potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs).
  • In some embodiments, a polypeptide of interest is a putative tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more putative tumor antigens. For example, members of a library include (e.g., internally express or carry) putative tumor antigens (e.g., a polypeptide previously identified (e.g., by a third party) as a tumor antigen, e.g., identified as a tumor antigen using a method other than a method of the present disclosure). In some embodiments, a putative tumor antigen is a tumor antigen described herein. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such putative tumor antigen mediates an immune response. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as stimulatory antigens. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as antigens that have minimal or no effect on an immune response. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as inhibitory and/or suppressive antigens.
  • In some embodiments, a polypeptide of interest is a pre-selected tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more pre-selected tumor antigens. For example, in some embodiments, members of a library include (e.g., internally express or carry) one or more polypeptides identified as tumor antigens using a method of the present disclosure and/or using a method other than a method of the present disclosure. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
  • In some embodiments, a polypeptide of interest is a known tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more known tumor antigens. For example, in some embodiments, members of a library include (e.g., internally express or carry) one or more polypeptides identified as a tumor antigen using a method of the present disclosure and/or using a method other than a method of the present disclosure. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein. In some embodiments, methods of the disclosure identify one or more known tumor antigens as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
  • In some embodiments, a polypeptide of interest is a potential tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more potential tumor antigens. For example, in some embodiments, members of a library include (e.g., internally express or carry) one or more polypeptides identified as being of interest, e.g., encoding mutations associated with a tumor, using a method of the present disclosure and/or using a method other than a method of the present disclosure. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such polypeptides mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein. In some embodiments, methods of the disclosure identify one or more polypeptides as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more polypeptides as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more polypeptides as inhibitory and/or suppressive antigens for one or more subjects.
    • Tumor Antigens
  • Polypeptides of interest used in methods and systems described herein include tumor antigens amd potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), and/or cancer/testis antigens (CTAs). Exemplary tumor antigens include, e.g., MART-1/MelanA (MART-I or MLANA), gp100 (Pmel 17 or SILV), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3 (also known as HIP8), BAGE, GAGE-1, GAGE-2, p15, Calcitonin, Calretinin, Carcinoembryonic antigen (CEA), Chromogranin, Cytokeratin, Desmin, Epithelial membrane protein (EMA), Factor VIII, Glial fibrillary acidic protein (GFAP), Gross cystic disease fluid protein (GCDFP-15), HMB-45, Human chorionic gonadotropin (hCG), inhibin, lymphocyte marker, MART-1 (Melan-A), Myo D1, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, PTPRC (CD45), S100 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor-1, Tumor M2-PK, vimentin, p53, Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens (e.g., EBNA1), human papillomavirus (HPV) antigen E6 or E7 (HPV_E6 or HPV_E7), TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1 (also known as CTAG1B), erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein (AFP), beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, MUC16, IL13Ra2, FRa, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, Nectin-4, AGS-16, guanalyl cyclase C, MUC-1, CFC1B, integrin alpha 3 chain (of a3b1, a laminin receptor chain), TPS, CD19, CD20, CD22, CD30, CD31, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CD34, CD99, CD117, CD80, CD28, CD13, CD15, CD25, CD10, CLL-1/CLEC12A, ROR1, Glypican 3 (GPC3), Mesothelin, CD33/IL3Ra, c-Met, PSCA, PSMA, Glycolipid F77, EGFRvIII, BCMA, GD-2, PSAP, prostein (also known as P501S), PSMA, Survivin (also known as BIRC5), and MAGE-A3, MAGEA2, MAGEA4, MAGEA6, MAGEA9, MAGEA10, MAGEA12, BIRC5, CDH3, CEACAM3, CGB isoform2, ELK4, ERBB2, HPSE1, HPSE2, KRAS_isoform1, KRAS_isoform2, MUC1, SMAD4, TERT.2. TERT.3, TGFBR2, EGAG9_isoform1, TP53, CGB isoforml, IMPDH2, LCK, angiopoietin-1 (Ang1) (also known as ANGPT1), XIAP (also known as BIRC4), galectin-3 (also known as LGALS3), VEGF-A (also known as VEGF), ATP6S1 (also known as ATP6AP1), MAGE-A1, cIAP-1 (also known as BIRC2), macrophage migration inhibitory factor (MIF), galectin-9 (also known as LGALS9), progranulin PGRN (also known as granulin), OGFR, MLIAP (also known as BIRC7), TBX4 (also known as ICPPS, SPS or T-Box4), secretory leukocyte protein inhibitor (Slpi) (also known as antileukoproteinase), Ang2 (also known as ANGPT2), galectin-1 (also known as LGALS1), TRP-2 (also known as DCT), hTERT (telomerase reverse transcriptase) tyrosinase-related protein 1 (TRP-1, TYRP1), NOR-90/UBF-2 (also known as UBTF), LGMN, SPA17, PRTN3, TRRAP_1, TRRAP_2, TRRAP_3, TRRAP_4, MAGEC2, PRAME, SOX10, RAC1, HRAS, GAGE4, AR, CYP1B1, MMP8, TYR, PDGFRB, KLK3, PAX3, PAXS, ST3GAL5, PLAC1, RhoC, MYCN, REG3A, CSAG2, CTAG2-1a, CTAG2-1b, PAGE4, BRAF, GRM3, ERBB4, KIT, MAPK1, MFI2, SART3, ST8SIA1, WDR46, AKAP-4, RGSS, FOSL1, PRM2, ACRBP, CTCFL, CSPG4, CCNB1, MSLN, WT1, SSX2, KDR, ANKRD30A, MAGED1, MAP3K9, XAGE1B, PREX2, CD276, TEK, AIM1, ALK, FOLH1, GRIN2A MAP3K5 and one or more isoforms of any preceding tumor antigens. Exemplary tumor antigens are provided in the accompanying list of sequences.
  • Tumor specific antigens (TSAs, or neoantigens) are tumor antigens that are not encoded in normal host genome (see, e.g., Yarchoan et al., Nat. Rev. Cancer. 2017 Feb. 24. doi: 10.1038/nrc.2016.154; Gubin et al., J. Clin. Invest. 125:3413-3421 (2015)). In some embodiments, TSAs arise from somatic mutations and/or other genetic alterations. In some embodiments, TSAs arise from missense or in-frame mutations. In some embodiments, TSAs arise from frame-shift mutations or loss-of-stop-codon mutations. In some embodiments, TSAs arise from insertion or deletion mutations. In some embodiments, TSAs arise from duplication or repeat expansion mutations. In some embodiments, TSAs arise from splice variants or improper splicing. In some embodiments, TSAs arise from gene fusions. In some embodiments, TSAs arise from translocations. In some embodiments, TSAs include oncogenic viral proteins. For example, as with Merkel cell carcinoma (MCC) associated with the Merkel cell polyomavirus (MCPyV) and cancers of the cervix, oropharynx and other sites associated with the human papillomavirus (HPV), TSAs include proteins encoded by viral open reading frames. For purposes of this disclosure, the terms “mutation” and “mutations” encompass all mutations and genetic alterations that may give rise to an antigen encoded in the genome of a cancer or tumor cell of a subject, but not in a normal or non-cancerous cell of the same subject. In some embodiments, TSAs are specific (personal) to a subject. In some embodiments, TSAs are shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1-10%, or more of subjects suffering from a cancer. In some embodiments, TSAs shared by more than one subject may be known or pre-selected.
  • In some embodiments, a TSA is encoded by an open reading frame from a virus. For example, a library can be designed to express polypeptides from one of the following viruses: an immunodeficiency virus (e.g., a human immunodeficiency virus (HIV), e.g., HIV-1, HIV-2), a hepatitis virus (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus, non-A and non-B hepatitis virus), a herpes virus (e.g., herpes simplex virus type I (HSV-1), HSV-2, Varicella-zoster virus, Epstein Barr virus, human cytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8), a poxvirus (e.g., variola, vaccinia, monkeypox, Molluscum contagiosum virus), an influenza virus, a human papilloma virus, adenovirus, rhinovirus, coronavirus, respiratory syncytial virus, rabies virus, coxsackie virus, human T cell leukemia virus (types I, II and III), parainfluenza virus, paramyxovirus, poliovirus, rotavirus, rhinovirus, rubella virus, measles virus, mumps virus, adenovirus, yellow fever virus, Norwalk virus, West Nile virus, a Dengue virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), bunyavirus, Ebola virus, Marburg virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Junin virus, Lassa virus, and Lymphocytic choriomeningitis virus. Libraries for other viruses can also be produced and used according to methods described herein.
  • Tumor specific antigens are known in the art, any of which can be used in methods described herein. In some embodiments, gene sequences encoding polypeptides that are potential or putative neoantigens are determined by sequencing the genome and/or exome of tumor tissue and healthy tissue from a subject having cancer using next generation sequencing technologies. In some embodiments, genes that are selected based on their frequency of mutation and ability to encode a potential or putative neoantigen are sequenced using next-generation sequencing technology. Next-generation sequencing applies to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChIP-sequencing), and epigenome characterization (de Magalhaes et al. (2010) Ageing Research Reviews 9 (3): 315-323; Hall N (2007) J. Exp. Biol. 209 (Pt 9): 1518-1525; Church (2006) Sci. Am. 294 (1): 46-54; ten Bosch et al. (2008) Journal of Molecular Diagnostics 10 (6): 484-492; Tucker T et al. (2009) The American Journal of Human Genetics 85 (2): 142-154). Next-generation sequencing can be used to rapidly reveal the presence of discrete mutations such as coding mutations in individual tumors, e.g., single amino acid changes (e.g., missense mutations, in-frame mutations) or novel stretches of amino acids generated by frame-shift insertions, deletions, gene fusions, read-through mutations in stop codons, duplication or repeat expansion mutations, and translation of splice variants or improperly spliced introns, and translocations (e.g., “neoORFs”).
  • Another method for identifying potential or putative neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn) including tandem mass spectrometry (MS/MS)) can also be used to identify neoantigens. Such proteomic approaches can be used for rapid, highly automated analysis (see, e.g., Gevaert et al., Electrophoresis 21:1145-1154 (2000)). High-throughput methods for de novo sequencing of unknown proteins can also be used to analyze the proteome of a subject's tumor to identify expressed potential or putative neoantigens. For example, meta shotgun protein sequencing may be used to identify expressed potential or putative neoantigens (see e.g., Guthals et al. (2012) Molecular and Cellular Proteomics 11(10):1084-96).
  • Potential or putative neoantigens may also be identified using MHC multimers to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples may be performed using MHC tetramer-based screening techniques (see e.g., Hombrink et al. (2011) PLoS One; 6(8): e22523; Hadrup et al. (2009) Nature Methods, 6(7):520-26; van Rooij et al. (2013) Journal of Clinical Oncology, 31:1-4; and Heemskerk et al. (2013) EMBO Journal, 32(2):194-203).
  • In some embodiments, one or more known or pre-selected tumor specific antigens, or one or more potential or putative tumor specific antigens identified using one of these methods, can be included in a library described herein.
  • Tumor associated antigens (TAAs) include proteins encoded in a normal genome (see, e.g., Ward et al., Adv. Immunol. 130:25-74 (2016)). In some embodiments, TAAs are either normal differentiation antigens or aberrantly expressed normal proteins. Overexpressed normal proteins that possess growth/survival-promoting functions, such as Wilms tumor 1 (WT1) (Ohminami et al., Blood 95:286-293 (2000)) or Her2/neu (Kawashima et al., Cancer Res. 59:431-435 (1999)), are TAAs that directly participate in the oncogenic process. Post-translational modifications, such as phosphorylation, of proteins may also lead to formation of TAAs (Doyle, J. Biol. Chem. 281:32676-32683 (2006); Cobbold, Sci. Transl. Med. 5:203ra125 (2013)). TAAs are generally shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1-10%, 1-20%, or more of subjects suffering from a cancer. In some embodiments, TAAs are known or pre-selected tumor antigens. In some embodiments, with respect to an individual subject, TAAs are potential or putative tumor antigens.Cancer/testis antigens (CTAs) are expressed by various tumor types and by reproductive tissues (for example, testes, fetal ovaries and trophoblasts) but have limited or no detectable expression in other normal tissues in the adult and are generally not presented on normal reproductive cells, because these tissues do not express MHC class I molecules (see, e.g., Coulie et al., Nat. Rev. Cancer 14:135-146 (2014); Simpson et al., Nat. Rev. Cancer 5:615-625 (2005); Scanlan et al., Immunol. Rev. 188:22-32 (2002)). Library Screens
  • Human Cells for Antigen Presentation
  • The present invention provides, inter alia, compositions and methods for identifying tumor antigens recognized by human immune cells. Human antigen presenting cells express ligands for antigen receptors and other immune activation molecules on human lymphocytes. Given differences in MHC peptide binding specificities and antigen processing enzymes between species, antigens processed and presented by human cells are more likely to be physiologically relevant human antigens in vivo than antigens identified in non-human systems. Accordingly, methods of identifying these antigens employ human cells to present candidate tumor antigen polypeptides. Any human cell that internalizes library members and presents polypeptides expressed by the library members on MHC molecules can be used as an antigen presenting cell according to the present disclosure. In some embodiments, human cells used for antigen presentation are primary human cells. The cells can include peripheral blood mononuclear cells (PBMC) of a human. In some embodiments, peripheral blood cells are separated into subsets (e.g., subsets comprising dendritic cells, macrophages, monocytes, B cells, or combinations thereof) prior to use in an antigen presentation assay. In some embodiments, a subset of cells that expresses MHC class II is selected from peripheral blood. In one example, a cell population including dendritic cells is isolated from peripheral blood. In some embodiments, a subset of dendritic cells is isolated (e.g., plasmacytoid, myeloid, or a subset thereof). Human dendritic cell markers include CD1c, CD1a, CD303, CD304, CD141, and CD209. Cells can be selected based on expression of one or more of these markers (e.g., cells that express CD303, CD1c, and CD141).
  • Dendritic cells can be isolated by positive selection from peripheral blood using commercially available kits (e.g., from Miltenyi Biotec Inc.). In some embodiments, the dendritic cells are expanded ex vivo prior to use in an assay. Dendritic cells can also be produced by culturing peripheral blood cells under conditions that promote differentiation of monocyte precursors into dendritic cells in vitro. These conditions typically include culturing the cells in the presence of cytokines such as GM-CSF and IL-4 (see, e.g., Inaba et al., Isolation of dendritic cells, Curr. Protoc. Immunol. May; Chapter 3: Unit 3.7, 2001). Procedures for in vitro expansion of hematopoietic stem and progenitor cells (e.g., taken from bone marrow or peripheral blood), and differentiation of these cells into dendritic cells in vitro, is described in U.S. Pat. No. 5,199,942, and U.S. Pat. Pub. 20030077263. Briefly, CD34+ hematopoietic stem and progenitor cells are isolated from peripheral blood or bone marrow and expanded in vitro in culture conditions that include one or more of Flt3-L, IL-1, IL-3, and c-kit ligand.
  • In some embodiments, immortalized cells that express human MHC molecules (e.g., human cells, or non-human cells that are engineered to express human MHC molecules) are used for antigen presentation. For example, assays can employ COS cells transfected with human MHC molecules or HeLa cells.
  • In some embodiments, both the antigen presenting cells and immune cells used in the method are derived from the same subject (e.g., autologous T cells and APC are used). In these embodiments, it can be advantageous to sequentially isolate subsets of cells from peripheral blood of the subject, to maximize the yield of cells available for assays. For example, one can first isolate CD4+ and CD8+ T cell subsets from the peripheral blood. Next, dendritic cells (DC) are isolated from the T cell-depleted cell population. The remaining T- and DC-depleted cells are used to supplement the DC in assays, or are used alone as antigen presenting cells. In some embodiments, DC are used with T- and DC-depleted cells in an assay, at a ratio of 1:2, 1:3, 1:4, or 1:5. In some embodiments, the antigen presenting cells and immune cells used in the method are derived from different subjects (e.g., heterologous T cells and APC are used).
  • Antigen presenting cells can be isolated from sources other than peripheral blood. For example, antigen presenting cells can be taken from a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, lymph nodes, spleen, bone marrow, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, or other tissue, for use in screening assays. In some embodiments, cells are taken from a tissue that is the site of an active immune response (e.g., an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
  • Antigen presenting cells useful in methods described herein are not limited to “professional” antigen presenting cells. In some embodiments, non-professional antigen presenting cells can be utilized effectively in the practice of methods of the present disclosure. Non-professional antigen presenting cells include fibroblasts, epithelial cells, endothelial cells, neuronal/glial cells, lymphoid or myeloid cells that are not professional antigen presenting cells (e.g., T cells, neutrophils), muscle cells, liver cells, and other types of cells.
  • Antigen presenting cells are cultured with library members that express a polypeptide of interest (and, if desired, a cytolysin polypeptide) under conditions in which the antigen presenting cells internalize, process and present polypeptides expressed by the library members on WIC molecules. In some embodiments, library members are killed or inactivated prior to culture with the antigen presenting cells. Cells or viruses can be inactivated by any appropriate agent (e.g., fixation with organic solvents, irradiation, freezing). In some embodiments, the library members are cells that express ORFs linked to a tag (e.g., a tag which comprises one or more known T cell epitopes) or reporter protein, expression of which has been verified prior to the culturing.
  • In some embodiments, antigen presenting cells are incubated with library members at 37° C. for between 30 minutes and 5 hours (e.g., for 45 min. to 1.5 hours). After the incubation, the antigen presenting cells can be washed to remove library members that have not been internalized. In certain embodiments, the antigen presenting cells are non-adherent, and washing requires centrifugation of the cells. The washed antigen presenting cells can be incubated at 37° C. for an additional period of time (e.g., 30 min. to 2 hours) prior to exposure to lymphocytes, to allow antigen processing. In some embodiments, it is desirable to fix and kill the antigen presenting cells prior to exposure to lymphocytes (e.g., by treating the cells with 1% paraformaldehyde).
  • The antigen presenting cell and library member numbers can be varied, so long as the library members provide quantities of polypeptides of interest sufficient for presentation on WIC molecules. In some embodiments, antigen presenting cells are provided in an array, and are contacted with sets of library cells, each set expressing a different polypeptide of interest. In certain embodiments, each location in the array includes 1×103-1×106 antigen presenting cells, and the cells are contacted with 1×103-1×108 library cells which are bacterial cells.
  • In any of the embodiments described herein, antigen presenting cells can be freshly isolated, maintained in culture, and/or thawed from frozen storage prior to incubation with library cells, or after incubation with library cells.
  • Human Lymphocytes
  • In methods of the present disclosure, human lymphocytes are tested for antigen-specific reactivity to antigen presenting cells, e.g., antigen presenting cells that have been incubated with libraries expressing polypeptides of interest as described above. The methods of the present disclosure permit rapid identification of human antigens using pools of lymphocytes isolated from an individual, or progeny of the cells. The detection of antigen-specific responses does not rely on laborious procedures to isolate individual T cell clones. In some embodiments, the human lymphocytes are primary lymphocytes. In some embodiments, human lymphocytes are NKT cells, gamma-delta T cells, or NK cells. Just as antigen presenting cells may be separated into subsets prior to use in antigen presentation assays, a population of lymphocytes having a specific marker or other feature can be used. In some embodiments, a population of T lymphocytes is isolated. In some embodiments, a population of CD4+ T cells is isolated. In some embodiments, a population of CD8+ T cells is isolated. CD8+ T cells recognize peptide antigens presented in the context of MHC class I molecules. Thus, in some embodiments, the CD8+ T cells are used with antigen presenting cells that have been exposed to library host cells that co-express a cytolysin polypeptide, in addition to a polypeptide of interest. T cell subsets that express other cell surface markers may also be isolated, e.g., to provide cells having a particular phenotype. These include CLA (for skin-homing T cells), CD25, CD30, CD69, CD154 (for activated T cells), CD45RO (for memory T cells), CD294 (for Th2 cells), γ/δ TCR-expressing cells, CD3 and CD56 (for NK T cells). Other subsets can also be selected.
  • Lymphocytes can be isolated, and separated, by any means known in the art (e.g., using antibody-based methods such as those that employ magnetic bead separation, panning, or flow cytometry). Reagents to identify and isolate human lymphocytes and subsets thereof are well known and commercially available.
  • Lymphocytes for use in methods described herein can be isolated from peripheral blood mononuclear cells, or from other tissues in a human. In some embodiments, lymphocytes are taken from tumors, lymph nodes, a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, spleen, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, peritoneal cavity, bone marrow, or other tissues. In some embodiments, cells are taken from a tissue that is the site of an active immune response (e.g., an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
  • Lymphocytes taken from an individual can be maintained in culture or frozen until use in antigen presentation assays. In some embodiments, freshly isolated lymphocytes can be stimulated in vitro by antigen presenting cells exposed to library cells as described above. In some embodiments, these lymphocytes exhibit detectable stimulation without the need for prior non-antigen specific expansion. However, primary lymphocytes also elicit detectable antigen-specific responses when first stimulated non-specifically in vitro. Thus, in some embodiments, lymphocytes are stimulated to proliferate in vitro in a non-antigen specific manner, prior to use in an antigen presentation assay. Lymphocytes can also be stimulated in an antigen-specific manner prior to use in an antigen presentation assay. In some embodiments, cells are stimulated to proliferate by a library (e.g., prior to use in an antigen presentation assay that employs the library). Expanding cells in vitro provides greater numbers of cells for use in assays. Primary T cells can be stimulated to expand, e.g., by exposure to a polyclonal T cell mitogen, such as phytohemagglutinin or concanavalin, by treatment with antibodies that stimulate proliferation, or by treatment with particles coated with the antibodies. In some embodiments, T cells are expanded by treatment with anti-CD2, anti-CD3, and anti-CD28 antibodies. In some embodiments, T cells are expanded by treatment with interleukin-2. In some embodiments, lymphocytes are thawed from frozen storage and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells. In some embodiments, lymphocytes are thawed from frozen storage and are not expanded prior to contacting with antigen presenting cells. In some embodiments, lymphocytes are freshly isolated and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells.
  • Antigen Presentation Assays
  • In antigen presentation assays, T cells are cultured with antigen presenting cells prepared according to the methods described above, under conditions that permit T cell recognition of peptides presented by MHC molecules on the antigen presenting cells. In some embodiments, T cells are incubated with antigen presenting cells at 37° C. for between 12-48 hours (e.g., for 24 hours). In some embodiments, T cells are incubated with antigen presenting cells at 37° C. for 3, 4, 5, 6, 7, or 8 days. Numbers of antigen presenting cells and T cells can be varied. In some embodiments, the ratio of T cells to antigen presenting cells in a given assay is 1:10, 1:5, 1:2, 1:1, 2:1, 5:1, 10:1, 20:1, 25:1, 30:1, 32:1, 35:1 or 40:1. In some embodiments, antigen presenting cells are provided in an array (e.g., in a 96-well plate), wherein cells in each location of the array have been contacted with sets of library cells, each set including a different polypeptide of interest. In certain embodiments, each location in the array includes 1×103-1×106 antigen presenting cells, and the cells are contacted with 1×103-1×106 T cells.
  • After T cells have been incubated with antigen presenting cells, cultures are assayed for activation. Lymphocyte activation can be detected by any means known in the art, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers. In some embodiments, culture supernatants are harvested and assayed for increased and/or decreased expression and/or secretion of one or more polypeptides associated with activation, e.g., a cytokine, soluble mediator, cell surface marker, or other immune mediator. In some embodiments, the one or more cytokines are selected from TRAIL, IFN-gamma, IL-12p′70, IL-2, TNF-alpha, MIP1-alpha, MIP1-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3-alpha, and fractalkine. In some embodiments, the one or more soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin. In some embodiments, the one or more cell surface markers are selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD1 (CD279), FoxP3, TIGIT, CD160, BTLA, 2B4 (CD244), and KLRG1. Cytokine secretion in culture supernatants can be detected, e.g., by ELISA, bead array, e.g., with a Luminex® analyzer. Cytokine production can also be assayed by RT-PCR of mRNA isolated from the T cells, or by ELISPOT analysis of cytokines released by the T cells. In some embodiments, proliferation of T cells in the cultures is determined (e.g., by detecting 3H thymidine incorporation). In some embodiments, target cell lysis is determined (e.g., by detecting T cell dependent lysis of antigen presenting cells labeled with Na2 51CrO4). Target cell lysis assays are typically performed with CD8+ T cells. Protocols for these detection methods are known. See, e.g., Current Protocols In Immunology, John E. Coligan et al. (eds), Wiley and Sons, New York, N.Y., 2007. One of skill in the art understands that appropriate controls are used in these detection methods, e.g., to adjust for non-antigen specific background activation, to confirm the presenting capacity of antigen presenting cells, and to confirm the viability of lymphocytes.
  • In some embodiments, antigen presenting cells and lymphocytes used in the method are from the same individual. In some embodiments, antigen presenting cells and lymphocytes used in the method are from different individuals.
  • In some embodiments, antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, e.g., to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g., to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, and antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g., to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using antigen presenting cells and lymphocytes from different individuals, e.g., to identify antigens recognized by multiple individuals, or compare reactivities that differ between individuals.
    • Methods of Identifying Tumor Antigens
  • One advantage of methods described herein is their ability to identify clinically relevant human antigens. Humans that have cancer may have lymphocytes that specifically recognize tumor antigens, which are the product of an adaptive immune response arising from prior exposure. In some embodiments, these cells are present at a higher frequency than cells from an individual who does not have cancer, and/or the cells are readily reactivated when re-exposed to the proper antigenic stimulus (e.g., the cells are “memory” cells). Thus, humans that have or have had cancer are particularly useful donors of cells for identifying antigens in vitro. The individual may be one who has recovered from cancer. In some embodiments, the individual has been recently diagnosed with cancer (e.g., the individual was diagnosed less than one year, three months, two months, one month, or two weeks, prior to isolation of lymphocytes and/or antigen presenting cells from the individual). In some embodiments, the individual was first diagnosed with cancer more than three months, six months, or one year prior to isolation of lymphocytes and/or antigen presenting cells.
  • In some embodiments, lymphocytes are screened against antigen presenting cells that have been contacted with a library of cells whose members express or carry polypeptides of interest, and the lymphocytes are from an individual who has not been diagnosed with cancer. In some embodiments, such lymphocytes are used to determine background (i.e., non-antigen-specific) reactivities. In some embodiments, such lymphocytes are used to identify antigens, reactivity to which exists in non-cancer individuals.
  • Cells from multiple donors (e.g., multiple subjects who have cancer) can be collected and assayed in methods described herein. In some embodiments, cells from multiple donors are assayed in order to determine if a given tumor antigen is reactive in a broad portion of the population, or to identify multiple tumor antigens that can be later combined to produce an immunogenic composition that will be effective in a broad portion of the population.
  • Antigen presentation assays are useful in the context of both infectious and non-infectious diseases. The methods described herein are applicable to any context in which a rapid evaluation of human cellular immunity is beneficial. In some embodiments, antigenic reactivity to polypeptides that are differentially expressed by neoplastic cells (e.g., tumor cells) is evaluated. Sets of nucleic acids differentially expressed by neoplastic cells have been identified using established techniques such as subtractive hybridization. Methods described herein can be used to identify antigens that were functional in a subject in which an anti-tumor immune response occurred. In other embodiments, methods are used to evaluate whether a subject has lymphocytes that react to a tumor antigen or set of tumor antigens.
  • In some embodiments, antigen presentation assays are used to examine reactivity to autoantigens in cells of an individual, e.g., an individual predisposed to, or suffering from, an autoimmune condition. Such methods can be used to provide diagnostic or prognostic indicators of the individual's disease state, or to identify autoantigens. For these assays, in some embodiments, libraries that include an array of human polypeptides are prepared. In some embodiments, libraries that include polypeptides from infectious agents which are suspected of eliciting cross-reactive responses to autoantigens are prepared. For examples of antigens from infectious agents thought to elicit cross-reactive autoimmune responses, see Barzilai et al., Curr Opin Rheumatol., 19(6):636-43, 2007; Ayada et al., Ann NY Acad Sci., 1108:594-602, 2007; Drouin et al., Mol Immunol., 45(1):180-9, 2008; and Bach, J Autoimmun., 25 Supp1:74-80, 2005.
  • As discussed, the present disclosure includes methods in which polypeptides of interest are included in a library (e.g., expressed in library cells or carried in or on particles or beads). After members of the library are internalized by antigen presenting cells, the polypeptides of interest are proteolytically processed within the antigen presenting cells, and peptide fragments of the polypeptides are presented on MEW molecules expressed in the antigen presenting cells. The identity of the polypeptide that stimulates a human lymphocyte in an assay described herein can be determined from examination of the set of library cells that were provided to the antigen presenting cells that produced the stimulation. In some embodiments, it is useful to map the epitope within the polypeptide that is bound by MEW molecules to produce the observed stimulation. This epitope, or the longer polypeptide from which it is derived (both of which are referred to as an “antigen” herein) can form the basis for an immunogenic composition, or for an antigenic stimulus in future antigen presentation assays.
  • Methods for identifying peptides bound by MEW molecules are known. In some embodiments, epitopes are identified by generating deletion mutants of the polypeptide of interest and testing these for the ability to stimulate lymphocytes. Deletions that lose the ability to stimulate lymphocytes, when processed and presented by antigen presenting cells, have lost the peptide epitope. In some embodiments, epitopes are identified by synthesizing peptides corresponding to portions of the polypeptide of interest and testing the peptides for the ability to stimulate lymphocytes (e.g., in antigen presentation assays in which antigen presenting cells are pulsed with the peptides). Other methods for identifying MEW bound peptides involve lysis of the antigen presenting cells that include the antigenic peptide, affinity purification of the MEW molecules from cell lysates, and subsequent elution and analysis of peptides from the MHC (Falk, K. et al. Nature 351:290, 1991, and U.S. Pat. No. 5,989,565).
  • In other embodiments, it is useful to identify the clonal T cell receptors that have been expanded in response to the antigen. Clonal T cell receptors are identified by DNA sequencing of the T cell receptor repertoire (Howie et al, 2015 Sci Trans Med 7:301). By identifying TCR specificity and function, TCRs can be transfected into other cell types and used in functional studies or for novel immunotherapies.
  • In other embodiments, it is useful to identify and isolate T cells responsive to a tumor antigen in a subject. The isolated T cells can be expanded ex vivo and administered to a subject for cancer therapy or prophylaxis.
    • Methods of Identifying Immune Responses of a Subject
  • The disclosure provides methods of identifying one or more immune responses of a subject (e.g., a test subject, or a target subject). In some embodiments, one or more immune responses of a subject (e.g., a test subject or a target subject) are determined by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the subject; c) contacting the antigen presenting cells with lymphocytes from the subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells. In some embodiments, the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more tumor antigens.
  • In some embodiments, a test subject is (i) a cancer subject who has not received a cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy; or (iii) a subject who has not been diagnosed with a cancer.
  • In some embodiments, a target subject is (i) a cancer subject who responds or has responded positively clinically (“responsive subject”) to a cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically (“non-responsive subject”) to a cancer therapy; (iii) a cancer subject who responds or has responded spontaneously to a cancer (“spontaneous target subject”); or (vi) a subject who has not been diagnosed with a cancer (“normal subject”).
  • In some embodiments, lymphocyte stimulation, non-stimulation, inhibition and/or suppression, activation, and/or non-responsiveness is determined by assessing levels of one or more expressed or secreted cytokines or other immune mediators described herein. In some embodiments, levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control indicates lymphocyte stimulation. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG). In some embodiments, a level of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level indicates lymphocyte inhibition and/or suppression. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level indicates lymphocyte inhibition and/or suppression. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control indicates lymphocyte inhibition and/or suppression. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG). In some embodiments, levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher or lower than a control level indicates lymphocyte activation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater or lower than the mean of a control level indicates lymphocyte activation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control indicates lymphocyte activation. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG). In some embodiments, a level of one or more expressed or secreted cytokines that is within about 20%, 15%, 10%, 5%, or less, of a control level indicates lymphocyte non-responsiveness or non-stimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is less than 1 or 2 standard deviations higher or lower than the mean of a control level indicates lymphocyte non-responsiveness or non-stimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is less than 1 or 2 median absolute deviations (MADs) higher or lower than a median response level to a control indicates lymphocyte non-responsiveness or non-stimulation. In some embodiments, a subject response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more cytokines) and of the total number of tumor antigens (e.g., of all or a portion of different tumor antigens from the library) that stimulate, do not stimulate, inhibit and/or suppress, activate, or have no or minimal effect on production, expression or secretion of each member of the panel of cytokines.
    • Method of Obtaining a Subject Response Profile
  • The disclosure provides methods for obtaining a subject response profile from a test subject (a “subject response profile”).
  • In some embodiments, the subject response profile of a test subject is obtained by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the test subject; c) contacting the antigen presenting cells with lymphocytes from the test subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells, to obtain the subject response profile. In some embodiments, the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000, or more tumor antigens.
  • The subject response profile can include a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, or to which lymphocytes are non-responsive. In some embodiments, the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • In some embodiments, the subject response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate expression or secretion of one or more immune mediators, that inhibit and/or suppress expression or secretion of one or more immune mediators, and/or which do not, or minimally, affect expression or secretion of immune mediators. In some embodiments, the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
    • Methods of Obtaining a Target Response Profile
  • In some embodiments, a subject response profile is compared to a corresponding response profile from a target subject, e.g. a cancer subject who responds and/or has responded clinically to a cancer therapy; a cancer subject who does not and/or has not responded clinically to a cancer therapy; a subject who has, or has had, spontaneous response to a cancer; or a subject who has not been diagnosed with a cancer (a “target response profile” of a target subject).
  • The disclosure provides methods for obtaining a target response profile from a target subject. The target response profile of a target subject is obtained by a) providing a library described herein that includes all or a portion of the same panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein) used to generate the subject response profile; b) contacting the library with antigen presenting cells from the target subject; c) contacting the antigen presenting cells with lymphocytes from the target subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to, one or more tumor antigens presented by one or more antigen presenting cells, to obtain the target response profile.
  • The target response profile includes a quantification, identification, and/or representation of the immune response of cells from the target subject to the same panel of tumor antigens included in the subject response profile.
  • In some embodiments, the target response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, and/or to which lymphocytes are non-responsive. In some embodiments, the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • In some embodiments, the target response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens identified by the methods of the disclosure, that stimulate expression and/or secretion of one or more immune mediators, that inhibit and/or suppress expression or secretion of one or more immune mediators, and/or which do not, or minimally, affect expression and/or secretion of immune mediators. In some embodiments, the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
    • Comparison of a Subject Response Profile to a Target Response Profile Lymphocytes
  • In some embodiments, a subject response profile is similar to the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that stimulate lymphocytes in the target response profile; if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that activate lymphocytes in the target response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the target response profile.
  • In some embodiments, a subject response profile is dissimilar from the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile; if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that activate lymphocytes in the target response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the target response profile.
  • In some embodiments, a subject response profile is similar to the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that stimulate lymphocytes in the target response profile; if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that activate lymphocytes in the target response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the target response profile.
  • In some embodiments, a subject response profile is dissimilar from the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%,or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile; and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile; if the identified tumor antigens that activate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that activate lymphocytes in the target response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the target response profile.
    • Cytokines
  • In some embodiments, the target response profile can include a quantification, identification, and/or representation of one or more cytokines and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on cytokine production, expression and/or secretion. In some embodiments, the target response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all) of the cytokines included in the subject response profile) and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on production, expression and/or secretion of the panel of cytokines.
  • In some embodiments, a subject response profile is similar to the target response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that inhibit and/or suppress one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that inhibit and/or suppress expression and/or secretion of the same one or more cytokines included in the target response profile; and/or if the total number of antigens that have no or minimal effect on expression and/or secretion of one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that that have no or minimal effect on the same one or more cytokines included in the target response profile.
  • In some embodiments, a subject response profile is dissimilar from the target response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile; if the total number of antigens that inhibit and/or suppress expression and/or secretion of one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in the target response profile; and/or if the total number of antigens that have no or minimal effect on expression and/or secretion of one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that that have no or minimal effect on the same one or more cytokines included in the target response profile.
  • The foregoing methods apply to subject response profiles and target response profiles obtained with libraries encoding polypeptides that are potential tumor antigens, as well as tumor antigens.
    • Methods of Identifying/Selecting Subjects for Cancer Therapy
  • The disclosure provides methods of identifying a test subject, e.g., a cancer subject, for initiation, continuation, modification, and/or discontinuation or in some cases non-initiation of a cancer therapy (e.g., a cancer therapy described herein). Generally, such methods include comparing one or more immune responses of a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy) to one or more immune responses of a target subject, who may be: (i) a cancer subject who responds or has responded positively clinically (“responsive subject”) to the cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically (“non-responsive subject”) to the cancer therapy; (iii) a cancer subject who responds or has responded spontaneously to a cancer (“spontaneous subject”); and/or (vi) a subject who has not been diagnosed with a cancer (“normal subject”).
  • One or more immune responses of the test subject that are the same or similar to one or more immune responses of a responsive subject and/or dissimilar to one or more immune responses of a non-responsive subject indicates that the test subject should initiate and/or continue and/or modify (e.g., increase and/or combine with one or more other modalities) the cancer therapy. One or more immune responses of the test subject that are dissimilar to one or more immune responses of a responsive subject and/or similar to (or same as) one or more immune responses of a non-responsive subject indicates that the cancer subject should not initiate and/or should discontinue and/or should modify (e.g., reduce and/or combine with one or more other modalities) the cancer therapy, and/or should initiate an alternative cancer therapy, or in some cases, no cancer therapy.
  • In some embodiments, a subject response profile that is similar to a target response profile (of a responsive subject) indicates the test subject should initiate and/or continue and/or modify (e.g., increase and/or combine with one or more other modalities) the cancer therapy. In some embodiments, methods described herein include selecting a test subject for initiation and/or continuation and/or modification (e.g., increase and/or combine with one or more other modalities) of the cancer therapy if the subject response profile is similar to a target response profile (of a responsive subject). In some embodiments, methods described herein include initiating and/or continuing and/or modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a responsive subject). In some embodiments, methods described herein include administering the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a responsive subject). In some embodiments, methods described herein include modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a responsive subject).
  • In some embodiments, a subject response profile that is dissimilar to a target response profile (of a responsive subject) indicates the test subject should not initiate and/or should modify (e.g., reduce and/or combine with one or more other modalities) and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy. In some embodiments, methods described herein include not selecting a test subject for initiation and/or selecting a test subject for modification (e.g., reduction and/or combination with one or more other modalities) and/or discontinuation of the cancer therapy and/or initiation of an alternative cancer therapy, if the subject response profile is dissimilar to a target response profile (of a responsive subject). In some embodiments, methods described herein include not initiating and/or modifying (e.g., reducing and/or combining with one or more other modalities) and/or discontinuing administration of the cancer therapy to a test subject and/or initiation of an alternative cancer therapy, if the subject response profile is dissimilar to a target response profile (of a responsive subject). In some embodiments, methods described herein include not administering the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a responsive subject). In some embodiments, methods described herein include modifying (e.g., reducing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a responsive subject). In some embodiments, methods described herein include administering an alternative cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a responsive subject).
  • In some embodiments, a subject response profile is compared to a corresponding response profile from a cancer subject who has not responded and/or is not responding and/or responds negatively, clinically to the cancer therapy (a “target response profile” of a non-responsive subject). In some embodiments, the target response profile (of a non-responsive subject) is obtained by providing a library described herein that includes all or a portion of the same panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein or identified using a method described herein) used to generate the subject response profile; contacting the library with antigen presenting cells from the non-responsive subject; contacting the antigen presenting cells with lymphocytes from the non-responsive subject; and determining whether one or more lymphocytes are stimulated, inhibited and/or suppressed by, or non-responsive to, one or more tumor antigens presented by one or more antigen presenting cells. The target response profile (of a non-responsive subject) includes a quantification, identification, and/or representation of the immune response of cells from the non-responsive cancer subject to the same panel of tumor antigens included in the subject response profile.
  • Methods for comparing a subject response profile to a target response profile, and parameters for determining similarity and dissimilarily of a subject response profile to a target response profile are provided in the disclosure.
  • In some embodiments, the target response profile (of a non-responsive subject) includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, and/or that inhibit and/or suppress lymphocytes. In some embodiments, a subject response profile is similar to the target response profile (of a nonresponsive subject) if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a nonresponsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that do not stimulate lymphocytes in the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a nonresponsive subject). In some embodiments, a subject response profile is dissimilar from the target response profile if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a nonresponsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a nonresponsive subject). In some embodiments, a subject response profile is similar to the target response profile (of a nonresponsive subject) if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a nonresponsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that do not stimulate lymphocytesin the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a non-responsive subject). In some embodiments, a subject response profile is dissimilar from the target response profile (of a non-responsive subject) if the identified tumor antigens that stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that stimulate lymphocytes in the target response profile (of a non-responsive subject); if the identified tumor antigens that do not stimulate lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that do not stimulate lymphocytes in the target response profile (of a nonresponsive subject); and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the target response profile (of a non-responsive subject).
  • In some embodiments, the target response profile (of a non-responsive subject) can include a quantification, identification, and/or representation of one or more cytokines and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, and/or inhibit and/or suppress cytokine production, expression and/or secretion. In some embodiments, the target response profile (of a nonresponsive subject) can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all), of the cytokines included in the subject response profile) and the total number of tumor antigens (e.g., of the same tumor antigens included in the subject response profile) that stimulate, do not stimulate, and/or inhibit and/or suppress production, expression and/or secretion of the panel of cytokines. In some embodiments, a subject response profile is similar to the target response profile (of a nonresponsive subject) if the total number of antigens that stimulate one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that stimulate the same one or more cytokines included in the target response profile (of a non-responsive subject); if the total number of antigens that do not stimulate one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile (of a nonresponsive subject); and/or if the total number of antigens that inhibit and/or suppress one or more cytokines included in the subject response profile differs by no more than 1, 2, 3, 4, 5,10, 15, 20, or 25 from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in the target response profile (of a non-responsive subject). In some embodiments, a subject response profile is dissimilar from the target response profile (of a non-responsive subject) if the total number of antigens that stimulate one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, or more, from the total number of antigens that stimulate the same one or more cytokines included in the target response profile (of a non-responsive subject); if the total number of antigens that not stimulate one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, or more, from the total number of antigens that do not stimulate the same one or more cytokines included in the target response profile (of a non-responsive subject); and/or if the total number of antigens that inhibit and/or suppress one or more cytokines included in the subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in the target response profile (of a non-responsive subject).
  • In some embodiments, a subject response profile that is dissimilar to a target response profile (of a non-responsive subject) indicates the test subject should initiate and/or continue and/or modify (e.g., increase and/or combine with one or more other modalities) the cancer therapy. In some embodiments, methods described herein include selecting a test subject for initiation and/or continuation and/or modification of (e.g., increasing and/or combining with one or more other modalities) the cancer therapy if the subject response profile is dissimilar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include initiating and/or continuing and/or modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include administering the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include modifying (e.g., increasing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is dissimilar to a target response profile (of a non-responsive subject).
  • In some embodiments, a subject response profile that is similar to a target response profile (of a non-responsive subject) indicates the test subject should not initiate, and/or should modify (e.g., reduce and/or combine with one or more other modalities), and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy. In some embodiments, methods described herein include not selecting a test subject for initiation and/or selecting a test subject for modification (e.g., reduction and/or combination with one or more other modalities) and/or discontinuation of the cancer therapy and/or initiation of an alternative cancer therapy, if the subject response profile is similar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include not initiating and/or modifying (e.g., reducing and/or combining with one or more other modalities) and/or discontinuing administration of the cancer therapy to a test subject and/or initiating an alternative cancer therapy, if the subject response profile is similar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include not administering the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include modifying (e.g., reducing and/or combining with one or more other modalities) administration of the cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a non-responsive subject). In some embodiments, methods described herein include administering an alternative cancer therapy to a test subject if the subject response profile is similar to a target response profile (of a non-responsive subject).
  • In some embodiments, a subject response profile described herein is compared to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) target response profiles of one or more responsive subjects and/or of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) non-responsive subjects. In some embodiments, a target response profile described herein (e.g., of a responsive subject or non-responsive subject) includes an average of one or more immune responses (described herein) from a population of responsive or non-responsive subjects, respectively. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) subject response profiles of the test subject are obtained (e.g., before, during, and/or after initiation, modification, and/or discontinuation of administration of the cancer therapy).
    • Methods of Selecting Tumor Antigens and Methods of Inducing an Immune Response in a Subject
  • In general, immune responses can be usefully defined in terms of their integrated, functional end-effects. Dhabar et al. (2014) have proposed that immune responses can be categorized as being immunoprotective, immunopathological, and immunoregulatory/inhibitory. While these categories provide useful constructs with which to organize ideas, an overall in vivo immune response is likely to consist of several types of responses with varying amounts of dominance from each category. Immunoprotective or beneficial responses are defined as responses that promote efficient wound healing, eliminate infections and cancer, and mediate vaccine-induced immunological memory. These responses are associated with cytokines and mediators such as IFN-gamma, IL-12, IL-2, Granzyme B, CD107, etc. Immunopathological or deleterious responses are defined as those that are directed against self (autoimmune disease like multiple sclerosis, arthritis, lupus) or innocuous antigens (asthma, allergies) and responses involving chronic, non-resolving inflammation. These responses can also be associated with molecules that are implicated in immunoprotective responses, but also include immune mediators such as TNF-alpha, IL-10, IL-13, IL-17, IL-4, IgE, histamine, etc. Immunoregulatory responses are defined as those that involve immune cells and factors that regulate (mostly down-regulate) the function of other immune cells. Recent studies suggest that there is an arm of the immune system that functions to inhibit immune responses. For example, regulatory CD4+CD25+FoxP3+ T cells, IL-10, and TGF-beta, among others have been shown to have immunoregulatory/inhibitory functions. The physiological function of these factors is to keep pro-inflammatory, allergic, and autoimmune responses in check, but they may also suppress anti-tumor immunity and be indicative of negative prognosis for cancer. In the context of tumors, the expression of co-stimulatory molecules often decreases, and the expression of co-inhibitory ligands increases. MEW molecules are often down-regulated on tumor cells, favoring their escape. The tumor micro-environment, including stromal cells, tumor associated immune cells, and other cell types, produce many inhibitory factors, such as, IL-10, TGF-β, and IDO. Inhibitory immune cells, including T regs, Tr1 cells, immature DCs (iDCs), pDCs, and MDSC can be found in the tumor microenvironment. (Y Li UT GSBS Thesis 2016). Examples of mediators and their immune effects are shown in Table 2.
  • TABLE 2
    Immune Mediators
    Beneficial Outcomes Deleterious Outcomes
    Cytokine Function Secreted by Cancer ID Al Cancer ID Al
    TRAIL Induces apoptosis of Most cells X X ? X ? ?
    tumor cells, induces
    immune suppressor
    cells
    IFN- Critical for innate T cells, NK X X ? X ? X
    gamma and adaptive cells, NKT
    immunity to cells
    pathogens, inhibits
    viral replication,
    increases MHC Class
    I expression
    IL-12 Th1 differentiation; DCs, macro- X X ? X ? X
    stimulates T cell phages,
    growth, induces IFN- neutron-
    gamma/TNF-alpha phils
    secretion from T
    cells, enhances CTLs
    IL-2 T cell proliferation, T cells, APCs X X X ? ? ?
    differentiation into
    effector and
    memory T cells and
    regulatory T cells
    TNF- Induces fevers, Macro- X X ? X ? X
    alpha apoptosis, phages,
    inflammation, APCs
    inhibits viral
    replication
    MIP-1 Chemotactic/pro- Macro- X X ? ? ? X
    alpha inflammatory phages, DCs,
    effects, activates T cells
    granulocytes,
    induces secretion of
    IL-1/IL6/TNF-alpha
    MIP-1 Chemotactic/pro- Macro- X X ? ? ? X
    beta inflammatory phages, DCs,
    effects, activates T cells
    granulocytes,
    induces secretion of
    IL-1/IL6/TNF-alpha
    CXCL9 T cell APCs X X ? X ? X
    chemoattractant,
    induced by IFN-gamma
    CXCL10 Chemoattractant for APCs X X ? ? ? X
    T cells,
    macrophages, NK
    and DCs, promotes T
    cell adhesion to
    endothelial cells
    MCP-1 Recruits monocytes, most cells X X ? X ? X
    memory T cells and
    DCS
    RANTES Recruits T cells, T cells X X ? ? ? X
    eosinophils,
    basophils, induces
    proliferation/activation
    of NK cells, T cell
    activation marker
    CXCL11 Chemoattractant for APCs X X ? ? ? X
    activated T cells
    IL-3 Stimulates T cells, APCs X X ? ? ? ?
    proliferation of
    myeloid cells,
    induces growth of T
    cells
    IL-17I Produced by Th17 T cells X X ? X ? X
    cells, induces
    production of IL6,
    GCSF, GMCSF, IL1b,
    TGF-beta, TNF-alpha,
    chemokines
    IL-18 Pro-inflammatory, Macro- X X ? X ? X
    induces cell- phages
    mediated immunity,
    production of IFN-
    gamma
    IL-21 Induces CD4 T cells X X X X ? ?
    proliferation,
    upregulated in
    Th2/Th17 TFh
    IL-22 Cell-mediated NK cells, T X X ? X ? X
    immunity, pro- cells
    inflammatory
    IL-23 Pro-inflammatory APCs X X ? X ? X
    IL-24 Controls survival and Monocytes- X X ? ? ? X
    proliferation macro-
    phages, Th2
    cells
    IL-27 Induces APCs, T cells X X X X ? X
    differentiation of T
    cells, upregulates IL-
    10, can be pro-or
    anti-inflammatory;
    promotes Th1/Tr1,
    inhibits Th2/Th17/
    regulatory T cells
    IL-32 Pro-inflammatory, T cells, NK X X ? X ? X
    increases secretion cells
    of inflammatory
    cytokines and
    chemokines
    CSF Induces myeloid APCs X X X ? ? ?
    cells to proliferate
    and differentiate
    GM-CSF Promotes T cells, X X ? ? ? X
    macrophage and macro-
    Eosinophil phages
    proliferation and
    maturation, growth
    factor
    TRANCE Helps DC T cells ? X ? X ? ?
    maturation/survival,
    T cell activation
    marker, anti-
    apoptotic, stimulates
    osteoclast activity
    MIP-3 Chemotactic for T X X ? ? ? X
    alpha cells, DCs
    fractalkine Chemotactic for T Endothelial X X ? ? ? X
    cells and monocytes cells
    IL-4 Stimulates B cells, Th2 cells, ? X ? X X X
    Th2 proliferation, basophils
    plasma cell
    differentiation, IgE,
    upregulates MHC
    Class II expression,
    decreases IFN-
    gamma production
    IL-10 Down regulates Th1 Monocytes X ? X X X X
    cytokines/MHC Class Th2 cells,
    II expression/Co- regulatory T
    stimulatory molecule cells
    expression
    IL-5 Stimulates B cells, Ig Th2 cells, ? X ? X X X
    secretion, eosinophil mast cells
    activation
    IL-13 Similar to IL4, Th2 cells, NK ? X ? X X X
    induces IgE cells, mast
    production, Th2 cells,
    cytokine eosinophils,
    basophils
    TGF-beta Inhibits T cell regulatory T ? ? X X X ?
    proliferation, cells
    activity, function;
    blocks effects of pro-
    inflammatory
    cytokines
    IL-1 beta Induces fevers, pro- Macro- X X ? X ? X
    inflammatory phages
    IL-6 Pro-inflammatory, T cells, ? X ? X X X
    drives osteoclast macro-
    formation, drives phages
    Th17
    IL-8 Recruits neutrophils Macro- ? X ? X ? X
    to site of infection phages,
    epithelial
    cells
    IL-31 Cell-mediated Th2 cells, X X ? X ? X
    immunity, pro- macro-
    inflammatory phages, DCs
    IL-15 T cell proliferation T cells, NK X X X ? ? ?
    and survival cells
    IL-9 Th2 proliferation, T cells, ? ? X X X ?
    cytokine secretion neutrophils,
    mast cells
    ID = Infectious disease
    IA = Autoimmune disease
  • In some embodiments, a tumor antigen stimulates one or more lymphocyte responses that are beneficial to the subject. In some embodiments, a tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to the subject. Examples of immune responses that may lead to beneficial anti-tumor responses include but are not limited to 1) cytotoxic CD8+ T cells which can effectively kill cancer cells and release the mediators perforin and/or granzymes to drive tumor cell death; and 2) CD4+ Th1 T cells which play an important role in host defense and can secrete IL-2, IFN-gamma and TNF-alpha. These are induced by IL-12, IL-2, and IFN gamma among other cytokines.
  • In some embodiments, a tumor antigen stimulates one or more lymphocyte responses that are deleterious or non-beneficial to the subject. In some embodiments, a tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are beneficial to the subject. Examples of immune responses that may lead to deleterious or non-beneficial anti-tumor responses include but are not limited to 1) T regulatory cells which are a population of T cells that can suppress an immune response and secrete immunosuppressive cytokines such as TGF-beta and IL-10 and express the molecules CD25 and FoxP3; and 2) Th2 cells which target responses against allergens but are not productive against cancer. These are induced by increased IL-4 and IL-10 and can secrete IL-4, IL-5, IL-6, IL-9 and IL-13.
  • The disclosure provides methods and systems for identifying and selecting tumor antigens. In some embodiments, methods and systems described herein can identify and select one or more tumor antigens to which one or more immune responses are stimulated in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy). In some embodiments, methods and systems described herein can identify and select one or more tumor antigens to which one or more immune responses are not stimulated in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy). In some embodiments, methods and systems described herein can identify and select one or more tumor antigens to which one or more immune responses are inhibited and/or suppressed in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy). In some embodiments, methods and systems described herein can identify and select one or more tumor antigens which elicit no or minimal immune responses in a cancer subject who has not received a cancer therapy (or who has not responded and/or is not responding, clinically to a cancer therapy).
  • In some embodiments, a composition comprising the one or more selected tumor antigens is administered to a cancer subject before, during, and/or after administration of a cancer therapy.
  • The disclosure provides methods for selecting tumor antigens identified by the methods herein based on comparison of a subject response profile to a target response profile. The disclosure also provides methods for selecting (or de-selecting) tumor antigens identified by the methods herein, based on association with desirable or beneficial responses. The disclosure also provides methods for selecting (or de-selecting) tumor antigens identified by the methods herein, based on association with undesirable, deleterious or non-beneficial responses. In some embodiments, the methods for selecting tumor antigens are combined. The methods may be combined in any order, e.g. selection may be carried out by comparison of a subject response profile to a target response profile, followed by selection based on association with a desirable (or undesirable) response; or, selection may be carried out based on association with a desirable (or undesirable) response, followed by comparison of the subject response profile to a target response profile.
  • Methods for identifying tumor antigens and potential tumor antigens are provided herein. Methods for generating or obtaining a subject response profile are provided herein. Methods for generating or obtaining a target response profile, e.g. a population-based or composite target response profile, are provided herein. Methods for comparison of a subject response profile to a target response profile are provided herein. Methods for determining whether a subject response profile is similar to a target response profile are provided herein.
  • In some embodiments, a subject response profile and target response profile are generated or obtained using the same plurality of polypeptides of interest. In some embodiments, a subject response profile and target response profile are generated or obtained using the same plurality of tumor antigens.
  • The target response profile includes a quantification, identification, and/or representation of one or more tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, and/or to which lymphocytes are non-responsive.
  • In some embodiments, one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the test subject (e.g., identified from the subject response profile), and the same one or more tumor antigens are identified as stimulating lymphocytes in the target subject (e.g., identified from the target response profile). In some embodiments, one or more tumor antigens are identified as stimulating lymphocytes in the test subject (e.g., identified from the subject response profile) and the same one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the target subject (e.g., identified from the target response profile). In some embodiments, one or more tumor antigens or potential tumor antigens are identified as eliciting minimal or no response from lymphocytes in the test subject (e.g., identified from the subject response profile), and the same one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing lymphocytes in the target subject (e.g., identified from the target response profile). In some embodiments, one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing, lymphocytes in the test subject (e.g., identified from the subject response profile), and the same one or more tumor antigens are identified as eliciting minimal or no response from lymphocytes in the target subject (e.g., identified from the target response profile).
  • Tumor antigens may be identified and/or selected on the basis of similarity or dissimilarity of a subject response profile to a target response profile. Tumor antigens may be identified and/or selected (or de-selected) based on association with desirable or beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on association with undesirable, deleterious or non-beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on a combination of the preceding methods, applied in any order.
    • All Positive Responders
  • In some embodiments, a subject response profile is compared to a corresponding response profile from a cancer subject who responds and/or has responded clinically to a cancer therapy (a “target response profile” of a responsive subject described herein). In some embodiments, a subject response profile is compared to a target response profile from a target subject who has not been diagnosed with cancer. In some embodiments, a subject response profile is compared to a target response profile from a target subject who has (or had) a beneficial response to cancer. In some embodiments, the subject has (or had) a positive clinical response to a cancer therapy or combination of therapies. In some embodiments, the subject had a spontaneous response to a cancer. In some embodiments, the subject is in partial or complete remission from cancer. In some embodiments, the subject has cleared a cancer. In some embodiments, the subject has not had a relapse, recurrence or metastasis of a cancer. In some embodiments, the subject has a positive cancer prognosis. In some embodiments, the subject has not experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • In some embodiments, one or more tumor antigens of the subject response profile which elicit responses that are different from, or dissimilar to, responses elicited by the same tumor antigens of the target response profile are selected. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with desirable or beneficial immune responses. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with undesirable, deleterious, or non-beneficial immune responses.
  • Responses whereby tumor antigens or immunogenic fragments thereof (i) stimulate lymphocyte responses that are beneficial to the subject, (ii) stimulate expression of cytokines that are beneficial to the subject, (iii) inhibit and/or suppress lymphocyte responses that are deleterious or non-beneficial to the subject, or (iv) inhibit and/or suppress expression of cytokines that are deleterious or non-beneficial to the subject, are termed “beneficial responses”.
  • In some embodiments, a selected tumor antigen stimulates one or more lymphocyte responses that are beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to the subject.
  • In some embodiments, a selected tumor antigen increases expression and/or secretion of cytokines that are beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses expression of cytokines that are deleterious or non-beneficial to the subject.
  • In some embodiments, administration of one or more selected tumor antigens to the subject elicits an immune response of the subject. In some embodiments, administration of one or more selected tumor antigens to the subject elicits a beneficial immune response of the subject. In some embodiments, administration of one or more selected tumor antigens to the subject elicits a beneficial response of the subject. In some embodiments, administration of one or more selected tumor antigens to the subject improves clinical response of the subject to a cancer therapy.
    • All Negative Responders
  • In some embodiments, a subject response profile is compared to a corresponding response profile from a cancer subject who does not respond and/or has not responded clinically to a cancer therapy (a “target response profile” of a non-responsive subject described herein). In some embodiments, a subject response profile is compared to a target response profile from a target subject who has (or had) a deleterious or non-beneficial response to cancer. In some embodiments, the subject has (or had) a negative clinical response to a cancer therapy or combination of therapies. In some embodiments, the subject has not cleared a cancer. In some embodiments, the subject has had a relapse, recurrence or metastasis of a cancer. In some embodiments, the subject has a negative cancer prognosis. In some embodiments, the subject has experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • Responses whereby tumor antigens or immunogenic fragments thereof (i) stimulate lymphocyte responses that are deleterious or not beneficial to the subject, (ii) stimulate expression of cytokines that are deleterious or not beneficial to the subject, (iii) inhibit and/or suppress lymphocyte responses that are beneficial to the subject, or (iv) inhibit and/or suppress expression of cytokines that are beneficial to the subject, are termed “deleterious or non-beneficial responses”.
  • In some embodiments, one or more tumor antigens of the subject response profile which elicit responses that are the same as, or similar to, responses elicited by the same tumor antigens of the target response profile are selected. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with desirable or beneficial immune responses. In some embodiments, one or more tumor antigens are selected (or de-selected) based on association with undesirable, deleterious, or non-beneficial immune responses.
  • In some embodiments, a selected tumor antigen stimulates one or more lymphocyte responses that are deleterious or non-beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses one or more lymphocyte responses that are beneficial to the subject.
  • In some embodiments, a selected tumor antigen increases expression and/or secretion of cytokines that are deleterious or non-beneficial to the subject. In some embodiments, a selected tumor antigen inhibits and/or suppresses expression of cytokines that are beneficial to the subject.
  • In some embodiments, the one or more tumor antigens are de-selected by the methods herein.
  • In some embodiments, the one or more selected tumor antigens are excluded from administration to a subject.
    • Methods of Selecting Potential Tumor Antigens
  • In well-established tumors, activation of endogenous anti-tumor T cell responses is often insufficient to result in complete tumor regression. Moreover, T cells that have been educated in the context of the tumor microenvironment sometimes are sub-optimally activated, have low avidity, and ultimately fail to recognize the tumor cells that express antigen. In addition, tumors are complex and comprise numerous cell types with varying degrees of expression of mutated genes, making it difficult to generate polyclonal T cell responses that are adequate to control tumor growth. As a result, researchers in the field have proposed that it is important in cancer subjects to identify the mutations that are “potential tumor antigens” in addition to those that are confirmed in the cancer subject to be recognized by their T cells.
  • There are currently no reliable methods of identifying potential tumor antigens in a comprehensive way. Computational methods have been developed in an attempt to predict what is an antigen, however there are many limitations to these approaches. First, modeling epitope prediction and presentation needs to take into account the greater than 12,000 HLA alleles encoding MHC molecules, with each subject expressing as many as 14 of them, all with different epitope affinities. Second, the vast majority of predicted epitopes fail to be found presented by tumors when they are evaluated using mass spectrometry. Third, the predictive algorithms do not take into account T cell recognition of the antigen, and the majority of predicted epitopes are incapable of eliciting T cell responses even when they are present. Finally, the second arm of cellular immunity, the CD4+ T cell subset, is often overlooked; the majority of in silico tools focus on MHC class I binders. The tools for predicting MHC class II epitopes are under-developed and more variable.
  • The present disclosure provides methods to a) identify polypeptides that are potential tumor antigens in antigen presentation assays of the disclosure, and b) select polypeptides on the basis of their antigenic potential. The methods are performed without making predictions about what could be a target of T cell responses or presented by MHC, and without the need for deconvolution. The methods can be expanded to explore antigenic potential in healthy subjects who share the same MHC alleles as a subject, to identify those potential tumor antigens that would be most suitable to include in an immunogenic composition or vaccine formulation. The methods ensure that the potential tumor antigen is processed and presented in the context of subject MHC molecules, and that T cells can respond to the potential tumor antigen if they are exposed to the potential tumor antigen under the right conditions (e.g., in the context of a vaccine with a strong danger signal from an adjuvant or delivery system).
  • The preceding methods for selection of tumor antigens may be applied to selection of potential tumor antigens, that is, polypeptides encoding one or more mutations present or expressed in a cancer or tumor cell of a subject.
  • Immunogenic Compositions and Uses thereof
  • The present disclosure provides compositions that include a tumor antigen or tumor antigens identified or selected by methods described herein, nucleic acids encoding the tumor antigens, and methods of using the compositions. In some embodiments, a composition includes tumor antigens that are peptides 8-40 amino acids, 8-60 amino acids, 8-100. 8-150,or 8-200 amino acids in length (e.g., MHC binding peptides, e.g., peptides 23-29, 24-28, 25-27, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-15, 8-12 amino acids in length). In some embodiments, a composition includes one or more tumor antigens that are about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the full-length polypeptides. In some embodiments, a composition includes one or more tumor antigens that are truncated by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids, relative to the full-length polypeptides. The compositions can include tumor antigens that are, or that comprise, MHC class I-binding peptides, MHC class II-binding peptides, or both MHC class I and MHC class II-binding peptides. Compositions can include a single tumor antigen, or multiple tumor antigens. In some embodiments, a composition includes a set of two, three, four, five, six, seven, eight, nine, ten, or more tumor antigens. In some embodiments, a composition includes ten, fifteen, twenty, twenty-five, thirty, or more tumor antigens. In some embodiments, the tumor antigens or peptides are provided as one or more fusion proteins. In some embodiments, a composition comprises nucleic acids encoding the tumor antigens or peptides. In some embodiments, the nucleic acids encoding the tumor antigens or peptides are provided as one or more fusion constructs.
  • The present disclosure provides immunogenic compositions comprising any combination of two or three TAAs: HPSE1 (SEQ ID NO: 6), HPSE2 (SEQ ID NO: 7), and/or SMAD4 (SEQ ID NO: 8).
  • HPSE encodes Heparinase, an endoglycosidase that cleaves heparan sulfate proteoglycans (HSPGs) into heparan sulfate side chains and core proteoglycans. HPSE participates in extracellular matrix (ECM) degradation and remodeling. There is a single functional heparinase: HPSE isoform 1 (HPSE1), a 543 amino acid protein. The splice variant HPSE isoform 2 (HPSE2) has no enzymatic activity, but may regulate HPSE1 activity. The active protein form of HPSE1 is a heterodimer of 8 and 50 kDa subunits which are non-covalently linked. The TIM barrel fold domain contains the active site, and the C-terminal domain of the protein is involved in nonenzymatic signaling and secretory functions. Potential T-cell epitopes within HPSE have been described (Tang. In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies. Cancer Sci 105 (2014) 9-17). The protein sequences of HPSE1 and HPSE2 may be found by searching in the publicly available database, UniProt (on the World Wide Web, at http://www.uniprot.org/uniprot/Q9Y251) and http://www.uniprot.org/uniprot/Q8WWQ2 respectively). The DNA sequence of HPSE1 and HPSE2 may be found by searching in the publicly available database, Entrez (on the World Wide Web https://www.ncbi.nlm.nih.gov/gene/10855 and https://www.ncbi.nlm.nih.gov/gene/60495 respectively).
  • SMAD4 encodes Mothers against decapentaplegic homolog 4, a signal transduction protein and tumor suppressor gene, which is a central mediator of downstream transcriptional output in TGFb signaling pathways. SMAD4 is a 552 amino acid, 60.4 KDa protein. SMAD4 exists as a monomer in the absence of TGF-beta activation, and a heterodimer on TGF-beta activation. SMAD4 is composed of two molecules of a C-terminally phosphorylated R-SMAD molecule, SMAD2 or SMAD3, and one molecule of SMAD4 to form the transcriptional active SMAD2/SMAD3-SMAD4 complex. SMAD4 regulates transcription of a number of target genes through binding to DNA, recognizing an 8-bp palindromic sequence (GTCTAGAC) called the Smad-binding element (SBE). The protein acts as a tumor suppressor and inhibits epithelial cell proliferation. The protein and DNA sequences of SMAD4 may be found by searching in the publicly available databases, UniProt and Entrez (on the World Wide Web, at http://www.uniprot.org/uniprot/Q13485 and https://www.ncbi.nlm.nih.gov/gene/4089 respectively).
  • The disclosure also provides nucleic acids encoding the tumor antigens. The nucleic acids can be used to produce expression vectors, e.g., for recombinant production of the tumor antigens, or for nucleic acid-based administration in vivo (e.g., DNA vaccination).
  • In some embodiments, tumor antigens are used in diagnostic assays. For these assays, compositions including the tumor antigens can be provided in kits, e.g., for detecting antibody reactivity, or cellular reactivity, in a sample from an individual.
  • In some embodiments, tumor antigen compositions are used to induce an immune response in a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The tumor antigen compositions can be used to raise antibodies (e.g., in a non-human animal, such as a mouse, rat, hamster, or goat), e.g., for use in diagnostic assays, and for therapeutic applications. For an example of a therapeutic use, a tumor antigen discovered by a method described herein may be a potent T cell and/or B cell antigen. Preparations of antibodies may be produced by immunizing a subject with the tumor antigen and isolating antiserum from the subject. Methods for eliciting high titers of high affinity, antigen-specific antibodies, and for isolating the tumor antigen-specific antibodies from antisera, are known in the art. In some embodiments, the tumor antigen compositions are used to raise monoclonal antibodies, e.g., human monoclonal antibodies.
  • In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject to provide a therapeutic response. In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject that redirects an undesirable immune response. In some embodiments, a tumor antigen composition elicits an immune response that causes the subject to have a positive clinical response described herein, e.g., as compared to a subject who has not been administered the tumor antigen composition. In some embodiments, a tumor antigen composition elicits an immune response that causes the subject to have an improved clinical response, e.g., as compared to a subject who has not been administered the tumor antigen composition. In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject for palliative effect. The response can be complete or partial therapy.
  • In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject to provide a prophylactic response. The response can be complete or partial protection.
  • In some embodiments, immunogenicity of a tumor antigen is evaluated in vivo. In some embodiments, humoral responses to a tumor antigen are evaluated (e.g., by detecting antibody titers to the administered tumor antigen). In some embodiments, cellular immune responses to a tumor antigen are evaluated, e.g., by detecting the frequency of antigen-specific cells in a sample from the subject (e.g., by staining T cells from the subject with WIC/peptide tetramers containing the antigenic peptide, to detect antigen-specific T cells, or by detecting antigen-specific cells using an antigen presentation assay such as an assay described herein). In some embodiments, the ability of a tumor antigen or antigens to elicit protective or therapeutic immunity is evaluated in an animal model. In some embodiments, the ability of a tumor antigen or antigens to stimulate or to suppress and/or inhibit immunity is evaluated in an animal model.
  • In some embodiments, the composition includes a pharmaceutically acceptable carrier or excipient. An immunogenic composition may also include an adjuvant for enhancing the immunogenicity of the formulation, (e.g., oil in water, incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, saponin adjuvants, toll-like receptor agonists, or muramyl dipeptides). Other adjuvants are known in the art.
  • In some embodiments, an immunogenic composition includes a tumor antigen linked to a carrier protein. Examples of carrier proteins include, e.g., toxins and toxoids (chemical or genetic), which may or may not be mutant, such as anthrax toxin, PA and DNI (PharmAthene, Inc.), diphtheria toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.) or CRM 197, tetanus toxin, tetanus toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.), tetanus toxin fragment Z, exotoxin A or mutants of exotoxin A of Pseudomonas aeruginosa, bacterial flagellin, pneumolysin, an outer membrane protein of Neisseria meningitidis (strain available from the ATCC (American Type Culture Collection, Manassas, Va.)), Pseudomonas aeruginosa Hcp1 protein, E. coli heat labile enterotoxin, shiga-like toxin, human LTB protein, a protein extract from whole bacterial cells, and any other protein that can be cross-linked by a linker. Other useful carrier proteins include high density lipoprotein (HDL), bovine serum albumin (BSA), P40, and chicken riboflavin. Many carrier proteins are commercially available (e.g., from Sigma Aldrich.).
  • In some embodiments, an immunogenic composition including a tumor antigen identified by a method described herein is used in conjunction with an available vaccine. For example, an antigen identified as described herein can be used as a supplemental component of a vaccine formulation, or as a boosting antigen in a vaccination protocol.
  • In some embodiments, an immunogenic composition is in a volume of about 0.5 mL for subcutaneous injection, 0.1 mL for intradermal injection, or 0.002-0.02 mL for percutaneous administration. A 0.5 ml dose of the composition may contain approximately 2-500 ug of the tumor antigen.
  • In some embodiments an immunogenic composition is administered parenterally (for instance, by subcutaneous, intramuscular, intravenous, or intradermal injection). In some embodiments, delivery by a means that physically penetrates the dermal layer is used (e.g., a needle, airgun, or abrasion).
  • In some embodiments, an immunogenic composition is administered to a subject, e.g., by intramuscular injection, intradermal injection, or transcutaneous immunization with appropriate immune adjuvants. Compositions can be administered, one or more times, often including a second administration designed to boost an immune response in a subject. The frequency and quantity of dosage of the composition can vary depending on the specific activity of the composition and clinical response of the subject, and can be determined by routine experimentation.
  • The formulations of immunogenic compositions can be provided in unit-dose or multi-dose containers, for example, sealed ampoules and vials and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier immediately prior to use.
    • Production of Tumor Antigens
  • A tumor antigen suitable for use in any method or composition of the disclosure may be produced by any available means, such as recombinantly or synthetically (see, e.g., Jaradat Amino Acids 50:39-68 (2018); Behrendt et al., J. Pept. Sci. 22:4-27 (2016)). For example, a tumor antigen may be recombinantly produced by utilizing a host cell system engineered to express a tumor antigen-encoding nucleic acid. Alternatively or additionally, a tumor antigen may be produced by activating endogenous genes. Alternatively or additionally, a tumor antigen may be partially or fully prepared by chemical synthesis.
  • Where proteins are recombinantly produced, any expression system can be used. To give but a few examples, known expression systems include, for example, E. coli, egg, baculovirus, plant, yeast, or mammalian cells.
  • In some embodiments, recombinant tumor antigen suitable for the present invention are produced in mammalian cells. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59,1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); Chinese hamster ovary cells+/−DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • In some embodiments, the present invention provides recombinant tumor antigen produced from human cells. In some embodiments, the present invention provides recombinant tumor antigen produced from CHO cells or HT1080 cells.
  • Typically, cells that are engineered to express a recombinant tumor antigen may comprise a transgene that encodes a recombinant tumor antigen described herein. It should be appreciated that the nucleic acids encoding recombinant tumor antigen may contain regulatory sequences, gene control sequences, promoters, non-coding sequences and/or other appropriate sequences for expressing the recombinant tumor antigen. Typically, the coding region is operably linked with one or more of these nucleic acid components.
  • The coding region of a transgene may include one or more silent mutations to optimize codon usage for a particular cell type. For example, the codons of a tumor antigen transgene may be optimized for expression in a vertebrate cell. In some embodiments, the codons of a tumor antigen transgene may be optimized for expression in a mammalian cell. In some embodiments, the codons of a tumor antigen transgene may be optimized for expression in a human cell.
    • Methods of Manufacturing Immunogenic Compositions
  • In some embodiments, the disclosure provides methods of manufacturing an immunogenic composition for administration to a subject in need thereof, the method comprising: a) providing, preparing, or obtaining a plurality of antigenic compositions comprising a plurality of antigens, each composition comprising a different antigen; b) providing, preparing, or obtaining a target response profile, wherein the target response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; c) providing, preparing, or obtaining a subject response profile, wherein the subject response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated (e.g., determined, measured, observed) with the plurality of antigens; d) comparing the target response profile to the subject response profile; e) selecting one or more antigens based on the comparison; and f) formulating at least a portion of one or more antigenic compositions comprising the one or more selected antigens as a pharmaceutical composition.
  • In some instances, about 1, 2, 5, 10, 20, 40, 60, 80, 100, 150, 200 or more, antigenic compositions are provided, prepared, or obtained. For example, a plurality of antigens can be produced using a method described herein, e.g., recombinantly or synthetically. The antigens can be provided in a suitable composition, such as a solution or lyophilized composition. In some instances, the antigens are synthetically produced. In some instances, a synthetically produced antigen remains attached to a solid support. In some instances, formulating an antigen includes aliquoting a portion of the antigenic composition, reconstituting at least a portion of a lyophilized antigenic composition, and/or releasing a synthetically produced antigen from a solid support.
  • Antigenic compositions may be prepared or obtained and stored in a variety of forms, such as in a suspension, in solution, or lyophilized. Antigenic compositions may be stored at a temperature ranging from less than −80° C. to about room temperature, for example at about −80° C., about −20° C., about −15° C., about −10° C., about 4° C. or at about room temperature. In some embodiments, antigenic compositions may include a carrier, excipient, stabilizer, preservative and/or adjuvant.
  • A plurality of antigens can be derived from a target response profile wherein the target response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated with (e.g., determined, measured, observed) with the plurality of antigens.
  • A plurality of antigens can be derived from a subject response profile wherein the subject response profile comprises a representation of the level of expression and/or secretion of one or more immune mediators associated with (e.g., determined, measured, observed) with the plurality of antigens.
  • In some embodiments, a target response profile and subject response profile are compared and one or more antigens are selected based on the comparision. In some embodiments, one or more antigens are selected that increase expression or secretion of immune mediators associated with a beneficial response to cancer, and/or one or more antigens that inhibit and/or suppress expression or secretion of immune mediators associated with deleterious or not beneficial responses to cancer. The selected antigens, or a portion of the selected antigens may be formulated as a pharmaceutical composition.
    • Cancer and Cancer Therapy
  • The present disclosure provides methods and systems related to subjects having or diagnosed with cancer, such as a tumor. In some embodiments, a tumor is or comprises a hematologic malignancy, including but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Langerhans cell histiocytosis, multiple myeloma, or myeloproliferative neoplasms.
  • In some embodiments, a tumor is or comprises a solid tumor, including but not limited to breast carcinoma, a squamous cell carcinoma, a colon cancer, a head and neck cancer, ovarian cancer, a lung cancer, mesothelioma, a genitourinary cancer, a rectal cancer, a gastric cancer, or an esophageal cancer.
  • In some particular embodiments, a tumor is or comprises an advanced tumor, and/or a refractory tumor. In some embodiments, a tumor is characterized as advanced when certain pathologies are observed in a tumor (e.g., in a tissue sample, such as a biopsy sample, obtained from a tumor) and/or when cancer patients with such tumors are typically considered not to be candidates for conventional chemotherapy. In some embodiments, pathologies characterizing tumors as advanced can include tumor size, altered expression of genetic markers, invasion of adjacent organs and/or lymph nodes by tumor cells. In some embodiments, a tumor is characterized as refractory when patients having such a tumor are resistant to one or more known therapeutic modalities (e.g., one or more conventional chemotherapy regimens) and/or when a particular patient has demonstrated resistance (e.g., lack of responsiveness) to one or more such known therapeutic modalities.
  • In some embodiments, the present disclosure provides methods and systems related to cancer therapy. The present disclosure is not limited to any specific cancer therapy, and any known or developed cancer therapy is encompassed by the present disclosure. Known cancer therapies include, e.g., administration of chemotherapeutic agents, radiation therapy, surgical excision, chemotherapy following surgical excision of tumor, adjuvant therapy, localized hypothermia or hyperthermia, anti-tumor antibodies, and anti-angiogenic agents. In some embodiments, cancer and/or adjuvant therapy includes a TLR agonist (e.g., CpG, Poly I:C, etc., see, e.g., Wittig et al., Crit. Rev. Oncol. Hematol. 94:31-44 (2015); Huen et al., Curr. Opin. Oncol. 26:237-44 (2014); Kaczanowska et al., J. Leukoc. Biol. 93:847-863 (2013)), a STING agonist (see, e.g., US20160362441; US20140329889; Fu et al., Sci. Transl. Med. 7:283ra52 (2015); and WO2014189805), a non-specific stimulus of innate immunity, and/or dendritic cells, or administration of GM-CSF, Interleukin-12, Interleukin-7, Flt-3, or other cytokines. In some embodiments, the cancer therapy is or comprises oncolytic virus therapy, e.g., talimogene leherparepvec. (see, e.g., Fukuhara et al., Cancer Sci. 107:1373-1379 (2016)). In some embodiments, the cancer therapy is or comprises bi-specific antibody therapy (e.g., Choi et al., 2011 Expert Opin Biol Ther; Huehls et al., 2015, Immunol and Cell Biol). In some embodiments, the cancer therapy is or comprises cellular therapy such as chimeric antigen receptor T (CAR-T) cells, TCR-transduced T cells, dendritic cells, tumor infiltrating lymphocytes (TIL), or natural killer (NK) cells (e.g., as reviewed in Sharpe and Mount, 2015, Dis Model Mech 8:337-50).
  • Anti-tumor antibody therapies (i.e., therapeutic regimens that involve administration of one or more anti-tumor antibody agents) are rapidly becoming the standard of care for treatment of many tumors. Antibody agents have been designed or selected to bind to tumor antigens, particularly those expressed on tumor cell surfaces. Various review articles have been published that describe useful anti-tumor antibody agents (see, for example, Adler et al., Hematol. Oncol. Clin. North Am. 26:447-81 (2012); Li et al., Drug Discov. Ther. 7:178-84 (2013); Scott et al., Cancer Immun. 12:14 (2012); and Sliwkowski et al., Science 341:1192-1198 (2013)). The below Table 8 presents a non-comprehensive list of certain human antigens targeted by known, available antibody agents, and notes c
  • Certain cancer indications for which the antibody agents have been proposed to be useful:
  • TABLE 8
    Human Antibody (commercial
    Antigen or scientific name) Cancer indication
    CD2 Siplizumab Non-Hodgkin's
    Lymphoma
    CD3 UCHT1 Peripheral or
    CD4 HuMax-CD4 Cutaneous
    T-cell Lymphoma
    CD19 SAR3419, MEDI-551 Diffuse Large B-cell
    Lymphoma
    CD19 and Bispecific antibodies Non-Hodgkin's
    CD3 or such as Blinatumomab, Lymphoma
    CD22 DT2219ARL
    CD20 Rituximab, Veltuzumab, B cell malignancies
    Tositumomab, (Non-Hodgkin's
    Ofatumumab, lymphoma, Chronic
    Ibritumomab, lymphocytic leukemia)
    Obinutuzumab,
    CD22 Inotuzumab, tetraxetan, Chemotherapy-resistant
    (SIGLEC2) CAT-8015, hairy cell leukemia,
    DCDT2980S, Hodgkin's lymphoma
    Bectumomab
    CD30 Brentuximab vedotin
    CD33 Gemtuzumab Acute myeloid
    ozogamicin leukemia
    (Mylotarg)
    CD37 16 Chronic lymphocytic
    leukemia
    CD38 mumab Multiple myeloma,
    hematological tumors
    CD40 mumab Non-Hodgkin's
    lymphoma
    CD52 Alemtuzumab Chronic lymphocytic
    (Campath) leukemia
    CD56 Lorvotuzumab Small Cell Lung
    (NCAM1) Cancer
    CD66e (CEA) Labetuzumab Breast, colon and
    lung tumors
    CD70 SGN-75 Non-Hodgkin's
    lymphoma
    CD74 Milatuzumab Non-Hodgkin's
    lymphoma
    CD138 (SYND1) BT062 Multiple Myeloma
    CD152 (CTLA-4) Ipilimumab Metastatic melanoma
    CD221 (IGF1R) AVE1642, IMC-A12, Glioma, lung, breast,
    MK-0646, head and neck,
    R150, CP 751871 prostate and thyroid
    cancer
    CD254 (RANKL) Denosumab Breast and prostate
    carcinoma
    CD261 (TRAILR1) Mapatumumab Colon, lung and
    CD262 (TRAILR2) HGS-ETR2, pancreas tumors and
    CS-1008 haematological
    malignancies
    CD326 (Epcam) Edrecolomab, Colon and rectal cancer,
    17-1A, IGN101, malignant ascites,
    Catumaxomab, epithelial tumors
    Adecatumumab (breast, colon, lung)
    CD309 (VEGFR2) IM-2C6, CDP791 Epithelium-derived
    solid tumors
    CD319 (SLAMF7) HuLuc63 Multiple myeloma
    CD340 (HER2) Trastuzumab, Breast cancer
    Pertuzumab, Ado-
    trastuzumab emtansine
    CAIX (CA9) cG250 Renal cell carcinoma
    EGFR (c-erbB) Cetuximab, Solid tumors including
    Panitumumab, glioma, lung, breast,
    nimotuzumab and 806 colon, and head and
    neck tumors
    EPHA3 (HEK) KB004, IIIA4 Lung, kidney and colon
    tumors, melanoma,
    glioma and
    haematological
    malignancies
    Episialin Epitumomab Epithelial ovarian
    tumors
    FAP Sibrotuzumab Colon, breast, lung,
    and F19 pancreas, and head and
    neck tumors
    HLA-DR beta Apolizumab Chronic lymphocytic
    leukemia, non-
    Hodkin's lymphoma
    FOLR-1 Farletuzumab Ovarian tumors
    5T4 Anatumomab Non-small cell lung
    cancer
    GD3/GD2 3F8, ch14.18, Neuroectodermal and
    KW-2871 epithelial tumors
    gpA33 huA33 Colorectal carcinoma
    GPNMB Glembatumumab Breast cancer
    HER3 (ERBB3) MM-121 Breast, colon, lung,
    ovarian, and prostate
    tumors
    Integrin αVβ3 Etaracizumab Tumor vasculature
    Integrin α5β1 Volociximab Tumor vasculature
    Lewis-Y antigen hu3S193, IgN311 Breast, colon, lung
    and prostate tumors
    MET (HGFR) AMG 102, METMAB, Breast, ovary and
    SCH900105 lung tumors
    Mucin-1/CanAg Pemtumomab, Breast, colon, lung
    oregovomab, and ovarian tumors
    Cantuzumab
    PSMA ADC, J591 Prostate Cancer
    Phosphatidylserine Bavituximab Solid tumors
    TAG-72 Minretumomab Breast, colon and
    lung tumors
    Tenascin 81C6 Glioma, breast and
    prostate tumours
    VEGF Bevacizumab Tumor vasculature
    PD-L1 Avelumab Non-small cell lung
    cancer, MCC
    CD274 Durvalumab Non-small cell
    lung cancer
    IDO enzyme IDO inhibitors Multiple
  • In some embodiments, a cancer therapy is or comprises immune checkpoint blockade therapy (see, e.g., Martin-Liberal et al., Cancer Treat. Rev. 54:74-86 (2017); Menon et al., Cancers (Basel) 8:106 (2016)), or immune suppression blockade therapy. Certain cancer cells thrive by taking advantage of immune checkpoint pathways as a major mechanism of immune resistance, particularly with respect to T cells that are specific for tumor antigens. For example, certain cancer cells may overexpress one or more immune checkpoint proteins responsible for inhibiting a cytotoxic T cell response. Thus, immune checkpoint blockade therapy may be administered to overcome the inhibitory signals and permit and/or augment an immune attack against cancer cells. Immune checkpoint blockade therapy may facilitate immune cell responses against cancer cells by decreasing, inhibiting, or abrogating signaling by negative immune response regulators (e.g., CTLA-4). In some embodiments, a cancer therapy or may stimulate or enhance signaling of positive regulators of immune response (e.g., CD28).
  • Examples of immune checkpoint blockade and immune suppression blockade therapy include agents targeting one or more of A2AR, B7-H4, BTLA, CTLA-4, CD28, CD40, CD137, GITR, IDO, KIR, LAG-3, PD-1, PD-L1, OX40, TIM-3, and VISTA. Specific examples of immune checkpoint blockade agents include the following monoclonal antibodies: ipilimumab (targets CTLA-4); tremelimumab (targets CTLA-4); atezolizumab (targets PD-L1); pembrolizumab (targets PD-1); nivolumab (targets PD-1); avelumab; durvalumab; and cemiplimab.
  • Specific examples of immune suppression blockade agents include: Vista (B7-H5, v-domain Ig suppressor of T cell activation) inhibitors; Lag-3 (lymphocyte-activation gene 3, CD223) inhibitors; IDO (indolemamine-pyrrole-2,3,-dioxygenase-1,2) inhibitors; KIR receptor family (killer cell immunoglobulin-like receptor) inhibitors; CD47 inhibitors; and Tigit (T cell immunoreceptor with Ig and ITIM domain) inhibitors.
  • In some embodiments, a cancer therapy is or comprises immune activation therapy. Specific examples of immune activators include: CD40 agonists; GITR (glucocorticoid-induced TNF-R-related protein, CD357) agonists; OX40 (CD134) agonists; 4-1BB (CD137) agonists; ICOS (inducible T cell stimulator); CD278 agonists; IL-2 (interleukin 2) agonists; and interferon agonists.
  • In some embodiments, cancer therapy is or comprises a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, or a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, and other cancer therapies.
  • As discussed herein, in some embodiments, the present disclosure provides methods and systems related to subjects who do not respond and/or have not responded; or respond and/or have responded (e.g., clinically responsive, e.g., clinically positively responsive or clinically negatively responsive) to a cancer therapy. In some embodiments, subjects respond and/or have responded positively clinically to a cancer therapy. In some embodiments, subjects respond and/or have responded negatively clinically to a cancer therapy. In some embodiments, subjects do not respond and/or have not responded (e.g., clinically non-responsive) to a cancer therapy.
  • Whether a subject responds positively, responds negatively, and/or fails to respond to a cancer therapy can be measured and/or characterized according to particular criteria. In certain embodiments, such criteria can include clinical criteria and/or objective criteria. In certain embodiments, techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MM, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology. A positive response, a negative response, and/or no response, of a tumor to a therapy can be assessed by ones skilled in the art using a variety of established techniques for assessing such response, including, for example, for determining one or more of tumor burden, tumor size, tumor stage, etc. Methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:e143-52.
  • In some embodiments, a responsive subject exhibits a decrease in tumor burden, tumor size, and/or tumor stage upon administration of a cancer therapy. In some embodiments, a non-responsive subject does not exhibit a decrease in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy. In some embodiments, a non-responsive subject exhibits an increase in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy.
  • In some embodiments, a cancer subject is identified and/or selected for administration of a cancer therapy as described herein. In some embodiments, the cancer therapy is administered to the subject. In some embodiments, upon administration of the cancer therapy, the subject exhibits a positive clinical response to the cancer therapy, e.g., exhibits an improvement based on one or more clinical and/or objective criteria (e.g., exhibits a decrease in tumor burden, tumor size, and/or tumor stage). In some embodiments, the clinical response is more positive than a clinical response to the cancer therapy administered to a cancer subject who is identified (using a method described herein) as a cancer subject who should not initiate, and/or should modify (e.g., reduce and/or combine with one or more other modalities), and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy.
  • Methods described herein can include preparing and/or providing a report, such as in electronic, web-based, or paper form. The report can include one or more outputs from a method described herein, e.g., a subject response profile described herein. In some embodiments, a report is generated, such as in paper or electronic form, which identifies the presence or absence of one or more tumor antigens (e.g., one or more stimulatory and/or inhibitory and/or suppressive tumor antigens, or tumor antigens to which lymphocytes are not responsive, described herein) for a cancer patient, and optionally, a recommended course of cancer therapy. In some embodiments, the report includes an identifier for the cancer patient. In one embodiment, the report is in web-based form.
  • In some embodiments, additionally or alternatively, a report includes information on prognosis, resistance, or potential or suggested therapeutic options. The report can include information on the likely effectiveness of a therapeutic option, the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to a cancer patient, e.g., identified in the report. For example, the report can include information, or a recommendation, on the administration of a cancer therapy, e.g., the administration of a pre-selected dosage or in a pre-selected treatment regimen, e.g., in combination with one or more alternative cancer therapies, to the patient. The report can be delivered, e.g., to an entity described herein, within 7, 14, 21, 30, or 45 days from performing a method described herein. In some embodiments, the report is a personalized cancer treatment report.
  • In some embodiments, a report is generated to memorialize each time a cancer subject is tested using a method described herein. The cancer subject can be reevaluated at intervals, such as every month, every two months, every six months or every year, or more or less frequently, to monitor the subject for responsiveness to a cancer therapy and/or for an improvement in one or more cancer symptoms, e.g., described herein. In some embodiments, the report can record at least the treatment history of the cancer subject.
  • In one embodiment, the method further includes providing a report to another party. The other party can be, for example, the cancer subject, a caregiver, a physician, an oncologist, a hospital, clinic, third-party payor, insurance company or a government office.
  • All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
  • The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.
    • EXAMPLES Example 1. Immune Responses to the ATLAS Melanoma Tumor Associated Antigen (TAA) Library—Single Patient Responses Generation of the ATLAS Melanoma TAA Library
  • 23 full-length genes (labelled as Un001-023, encoding known TAAs as shown below in Table 3) were obtained from the DNA Resource Core at Harvard Medical School, recloned into the ATLAS expression vector (Genocea Biosciences), and sequence-verified. Each TAA was recombinantly expressed in E. coli. Protein expression was verified using a surrogate T cell assay (the B3Z hybridoma) which recognizes the C57BL/6 mouse T cell epitope SIINFEKL (SEQ ID NO: 452), which is inserted at the C-terminus of each open reading frame, upstream of the stop codon. Proteins that induced B3Z responses that exceeded 5% of the positive control (the minimal SIINFEKL (SEQ ID NO: 452) epitope pulsed onto antigen presenting cells) were considered expressed.
  • TABLE 3
    ATLAS melanoma TAA library
    Antigen
    Code Name Alias Long Name OMIM GeneID
    Un001 MAGEA3 HIP8; HYPD; MAGE family member A3 300174 4102
    CT1.3;
    MAGE3;
    MAGEA6,
    MAGE-A3
    (G-2544)
    Un002 NY-ESO-1 CTAG; ESO1; cancer/testis antigen 1B 300156 1485
    CT6.1; CTAG1;
    LAGE-2;
    LAGE2B; NY-
    ESO-1
    Un003 ANGPT1 AGP1, AGPT, Angiopoietin-1 601667 284
    ANG1
    Un004 XIAP API3; ILP1; X-linked inhibitor of 300079 331
    MIHA; XLP2; apoptosis
    BIRC4; IAP-3;
    hIAP3; hIAP-3
    Un005 LGALS3 L31; GAL3; Galectin-3 153619 3958
    MAC2; CBP35;
    GALBP;
    GALIG;
    LGALS2
    Un006 VEGF-A VPF; VEGF; vascular endothelial growth 192240 7422
    MVCD1 factor A
    Un007 ATP6AP1 16A; CF2; ATPase H+ transporting 300197 537
    Ac45; XAP3; accessory protein 1
    XAP-3;
    ATP6S1;
    VATPS1;
    ATP6IP1
    Un008 MAGEA1 CT1.1; MAGE1 MAGE family member A1 300016 4100
    Un009 BIRC2 API1; MIHB; baculoviral IAP repeat 601712 329
    HIAP2; RNF48; containing 2
    cIAP1; Hiap-2;
    c-IAP1
    Un010 MIF GIF; GLIF; Macrophage migration 153620 4282
    MMIF, MIF inhibitory factor
    Un011 LGALS9 HUAT; Galectin-9 601879 3965
    LGALS9A
    Un012 PMEL P1; SI; SIL; premelanosome protein 155550 6490
    ME20; P100;
    SILV; ME20M;
    gp100; ME20-
    M; PMEL17;
    D12S53E
    Un013 GRN GEP; GP88; Progranulin 138945 2896
    PEPI; PGRN;
    CLN11;
    PCDGF
    Un014 OGFR opioid growth factor receptor 606459 11054
    Un015 BIRC5 API4; EPR-1; Surviving 603352 332
    survivin, BIRC5
    Un016 BIRC7 KIAP; LIVIN; baculoviral IAP repeat 79444
    MLIAP; containing 7
    RNF50; ML-
    IAP
    Un017 TBX4 SPS T-box 4 601719 9496
    Un018 SLPI ALP; MPI; Secretory leukocyte protein 107285 6590
    ALK1; BLPI; inhibitor
    HUSI; WAP4;
    WFDC4; HUSI-
    I
    Un019 ANGPT2 AGPT2, ANG2 Angiopoietin-2 601922 285
    Un020 LGALS1 GBP; GAL1 Galectin-1 150570 3956
    Un021 DCT TRP-2; TYRP2, , dopachrome tautomerase 191275 1638
    Un100 TYRP-2
    Un022 MLANA MART1; Melan-A 605513 2315
    MART-1
    Un023 TERT TP2; TRT; telomerase reverse 187270 7015
    CMM9; EST2; transcriptase
    TCS1; hTRT;
    DKCA2;
    DKCB4;
    hEST2;
    PFBMFT1
    Un024 LGMN AEP; LGMN1; legumain 602620 5641
    PRSC1
    Un025 SPA17 CT22; SP17; sperm surface protein Sp17 608621 53340
    SP17-1
    Un026 HPV_E7 HPV E7 oncoprotein 1489079
    Un027 TP53 P53; BCC7; cellular tumor antigen p53 191170 7157
    LFS1; TRP53
    Un028 CEACAM3 CEA; CGM1; carcinoembryonic antigen- 609142 1084
    W264; W282; related cell adhesion molecule
    CD66D 3
    Un029 PRTN3 MBN; MBT; myeloblastin precursor 177020 5657
    NP4; P29; PR3;
    ACPA; AGP7;
    NP-4; PR-3;
    CANCA; C-
    ANCA
    Un030 TRRAP PAF350/400, transformation/transcription 603015 8295
    Un031 PAF400, domain associated protein
    Un046 STAF40, TR-
    Un092 AP, Tra1
    Un032 MAGEA12 CT1.12; melanoma-associated antigen 300177 4111
    MAGE12 12
    Un033 MAGEA2 CT1.2; melanoma-associated antigen 300173 4101
    MAGE2; 2
    MAGEA2A
    Un034 MAGEA9 CT1.9; MAGE9 melanoma-associated antigen 300342 4108
    9
    Un035 MAGEC2 CT10; melanoma-associated antigen 300468 51438
    HCA587; C2
    MAGEE1
    Un036 PRAME MAPE; OIP4; melanoma antigen 606021 23532
    CT130; O1P-4 preferentially expressed in
    tumors
    Un037 SOX10 DOM; WS4; transcription factor SOX-10 602229 6663
    PCWH; WS2E;
    WS4C
    Un038 MUC1 EMA; MCD; mucin-1 158340 4582
    PEM; RUM;
    KL-6; MAM6;
    MCKD; PEMT;
    CD227;
    H23AG;
    MCKD1; MUC-
    1; ADMCKD;
    ADMCKD1;
    CA 15-3; MUC-
    1/X;
    MUC1/ZD;
    MUC-1/SEC
    Un039 RAC1 MIG5; Rac-1; ras-related C3 botulinum 602048 5879
    TC-25; p21- toxin substrate 1 isoform
    Rac1 Rac1b
    Un040 HRAS CTLO; GTPase HRas 190020 3265
    HAMSV;
    HRAS1;
    RASH1; p21ras;
    C-H-RAS; H-
    RASIDX; C-
    BAS/HAS; C-
    HA-RAS1
    Un041 GAGE4 CT4.4 G antigen 12I 300597 2576
    Un042 BAGE BAGE1; CT2.1 B melanoma antigen 1 605167 574
    precursor
    Un043 AR KD; AIS; AR8; androgen receptor 313700 367
    TFM; DHTR;
    SBMA;
    HYSP1;
    NR3C4;
    SMAX1;
    HUMARA
    Un044 CYP1B1 CP1B; GLC3A; cytochrome P450 1B1 601771 1545
    CYPIB1;
    P4501B1
    Un045 CA9 MN; CAIX carbonic anhydrase 9 603179 768
    precursor
    Un047 MMP8 HNC; CLG1; neutrophil collagenase 120355 4317
    MMP-8;
    PMNL-CL
    Un048 GAGE1 CT4.1; GAGE-1 G antigen 1 300594 2543
    Un049 TYR ATN; CMM8; tyrosinase precursor 606933 7299
    OCA1;
    OCA1A;
    OCAIA; SHEP3
    Un050 HPV_E6 HPV E6 oncoprotein 1489078
    Un051 Bcr-abl BCR/ABL fusion protein 107963955
    e14a5, (peptide atlas
    A6MFJ9)
    Un052 PDGFRB IMF1; IBGC4; platelet-derived growth factor 173410 5159
    JTK12; receptor beta
    PDGFR;
    CD140B;
    PDGFR1;
    PDGFR-1
    Un053 KLK3 KLKB1, KLK3, Plasma kallikrein 176820 354
    PSA
    Un054 PAX5 ALL3; BSAP paired box protein Pax-5 167414 5079
    Un055 ST3GAL5 SATI; SIAT9; lactosylceramide alpha-2,3- 604402 8869
    ST3GalV; sialyltransferase
    SIATGM3S
    Un056 PLAC1 CT92; OOSP2L placenta-specific protein 1 300296 10761
    precursor
    Un057 PSCA PRO232 prostate stem cell antigen 602470 8000
    preproprotein
    Un058 RhoC H9; ARH9; rho-related GTP-binding 165380 389
    ARHC; RHOH9 protein RhoC precursor
    Un059 MYCN NMYC; ODED; N-myc proto-oncogene 164840 4613
    MODED; N- protein
    myc; bHLHe37
    Un060 EpCAM ESA; KSA; epithelial cell adhesion 185535 4072
    M4S1; MK-1; molecule
    DIAR5; EGP-2;
    EGP40; KS1/4;
    MIC18;
    TROP1;
    EGP314;
    HNPCC8;
    TACSTD1
    Un061 REG3A HIP; PAP; regenerating islet-derived 167805 5068
    PAP1; REG3; protein 3-alpha precursor
    INGAP; PAP-
    H; PBCGF;
    HIP/PAP; REG-
    III
    Un062 EphA2 ECK; CTPA; ephrin type-A receptor 2 176946 1969
    ARCC2;
    CTPP1;
    CTRCT6
    Un063 CSAG2 TRAG3; chondrosarcoma-associated 102723547
    CSAG3B; gene 2/3 protein
    CT24.2; TRAG-
    3
    Un064 CTAG2-1a CT2; ESO2; cancer/testis antigen 2 isoform 30848
    CAMEL; LAGE-1a
    CT6.2; CT6.2a;
    CT6.2b; LAGE-
    1; LAGE2B
    Un065 PAGE4 JM27; JM-27; P antigen family member 4 300287 9506
    CT16.7; GAGE-
    9; GAGEC1;
    PAGE-1;
    PAGE-4
    Un066 BRAF NS7; BRAF1; serine/threonine-protein 164757 673
    RAFB1; B- kinase B-raf
    RAF1
    Un067 FAP FAPA; SIMP; prolyl endopeptidase FAP 600403 2191
    DPPIV
    Un068 GRM3 GLUR3; metabotropic glutamate 601115 2913
    mGlu3; receptor 3
    GPRC1C;
    MGLUR3
    Un069 ERBB4 HER4; ALS19; receptor tyrosine-protein 600543 2066
    p180erbB4 kinase erbB-4
    Un070 KIT PBT; SCFR; C- mast/stem cell growth factor 164920 3815
    Kit; CD117 receptor Kit
    Un071 LCK LSK; YT16; tyrosine-protein kinase Lck 153390 3932
    IMD22; p56lck;
    pp58lck
    Un072 MAGEA10 CT1.10; melanoma-associated antigen 300343 4109
    MAGE10 10
    Un073 MAGEA4 CT1.4; melanoma-associated antigen 300175 4103
    MAGE4; 4
    MAGE4A;
    MAGE4B;
    MAGE-41;
    MAGE-X2
    Un074 MAGEA6 CT1.6; melanoma-associated antigen 300176 4105
    MAGE6; 6
    MAGE3B;
    MAGE-3b
    Un075 MAPK1 ERK; p38; p40; mitogen-activated protein 176948 5594
    p41; ERK2; kinase 1
    ERT1; ERK-2;
    MAPK2;
    PRKM1;
    PRKM2;
    P42MAPK;
    p41mapk; p42-
    MAPK
    Un076 MFI2 MTf; MTF1; melanotransferrin 155750 4241
    CD228; MAP97
    Un077 SART3 P100; p110; squamous cell carcinoma 611684 9733
    DSAP1; antigen recognized by T-cells
    TIP110; 3
    p110(nrb);
    RP11-13G14
    Un078 ST8SIA1 GD3S; SIAT8; alpha-N-acetylneuraminide 601123 6489
    SIAT8A; alpha-2,8-sialyltransferase
    SIAT8-A;
    ST8SiaI
    Un079 WDR46 UTP7; BING4; WD repeat-containing protein 611440 9277
    FP221; C6orf11 46
    Un080 AKAP-4 AKAP 82, A-kinase anchoring protein 4 300185 8852
    AKAP-4,
    AKAP82,
    CT99, FSC1,
    HI, PRKA4,
    hAKAP82, p8,
    AKAP4
    Un081 RGS5 MST092; regulator of G-protein 603276 8490
    MST106; signaling 5
    MST129;
    MSTP032;
    MSTP092;
    MSTP106;
    MSTP129
    Un082 CTAG2-1b CT2; ESO2; cancer/testis antigen 2 isoform
    CAMEL; LAGE-1b
    CT6.2; CT6.2a;
    CT6.2b; LAGE-
    1; LAGE2B
    Un083 FOSL1 FRA; FRA1; fos-related antigen 1 136515 8061
    fra-1
    Un084 PRM2 MAD-CT-1; protamine-2 182890 5620
    CT94.2
    Un085 ACRBP CT23; SP32; acrosin-binding protein 608352 84519
    OY-TES-1 precursor
    Un086 AFP AFPD, FETA, alpha-fetoprotein 104150 174
    HPAFP
    Un087 CTCFL CT27; BORIS; transcriptional repressor 607022 140690
    CTCF-T; CTCFL
    HMGB1L1;
    dJ579F20.2
    Un088 CSPG4 NG2; MCSP; chondroitin sulfate 601172 1464
    MCSPG; proteoglycan 4 precursor
    MSK16; HMW-
    MAA; MEL-
    CSPG
    Un089 PAX3 WS1; WS3; paired box protein Pax-3 606597 5077
    CDHS; HUP2
    Un090 CCNB1 CCNB G2/mitotic-specific cyclin-B1 123836 891
    Un091 MSLN Mesotheline; mesothelin 601051 10232
    MPF; SMRP
    Un093 EGFR ERBB; HER1; epidermal growth factor 131550 1956
    mENA; receptor
    ERBB1; PIG61;
    NISBD2
    Un094 WT1 GUD; AWT1; Wilms tumor protein 607102 7490
    WAGR; WT33;
    NPHS4; WIT-2;
    EWS-WT1
    Un095 SSX2 SSX; HD21; protein SSX2 300192 6757
    CT5.2; CT5.2A;
    HOM-MEL-40
    Un096 KDR FLK1; CD309; vascular endothelial growth 191306 3791
    VEGFR; factor receptor 2 precursor
    VEGFR2
    Un097 ANKRD30A NY-BR-1 ankyrin repeat domain- 610856 91074
    containing protein 30A
    Un098 MAGED1 NRAGE; melanoma-associated antigen 300224 9500
    DLXIN-1 D1
    Un099 CEACAM5 CEA; CD66e carcinoembryonic antigen- 114890 1048
    related cell adhesion molecule
    5
    Un101 MAP3K9 MLK1; mitogen-activated protein 600136 4293
    MEKK9; kinase kinase kinase 9
    PRKE1
    Un102 XAGE1B CTP9; XAGE1; X antigen family member 1 300742 653220
    CT12.1;
    GAGED2;
    XAGE-1;
    XAGE1A;
    CT12.1A;
    CT12.1B
    Un103 PREX2 DEP.2; phosphatidylinositol 3,4,5- 612139 80243
    DEPDC2; P- trisphosphate-dependent Rac
    REX2; exchanger 2 protein
    PPP1R129;
    6230420N16Rik
    Un104 ERBB2 NEU; NGL; receptor tyrosine-protein 164870 2064
    HER2; TKR1; kinase erbB-2
    CD340; HER-2;
    MLN 19; HER-
    2/neu
    Un105 CD276 B7H3; B7-H3; CD276 antigen 605715 80381
    B7RP-2; 4Ig-
    B7-H3
    Un106 TEK TIE2; VMCM; angiopoietin-1 receptor 600221 7010
    TIE-2;
    VMCM1;
    CD202B
    Un107 AIM1 ST4; CRYBG1 absent in melanoma 1 protein 601797 202
    Un108 ALK CD246; ALK tyrosine kinase receptor 613014 238
    NBLST3
    Un109 PSMA FOLH1 Glutamate carboxypeptidase 2 600934 2346
    Un110 GRIN2A LKS; EPND; glutamate receptor ionotropic, 138253 2903
    FESD; NR2A; NMDA 2A
    GluN2A;
    NMDAR2A
    Un111 MAP3K5 ASK1; mitogen-activated protein 602448 4217
    MEKK5; kinase kinase kinase 5
    MAPKKK5
    Un112 HPSE1 heparanase isoform 1 604724 10855
    Un113 HPSE2 heparanase isoform 2 604724 10855
    Un114 SAGE CT14 sarcoma antigen 1 300359 55511
    OMIM = Online Mendelian Inheritance in Man database
    GeneID = NCBI database
    • ATLAS Library Screening
  • A peripheral blood sample was collected from a consented melanoma patient who had previously undergone therapy with a checkpoint inhibitor (pembrolizumab) and responded to therapy. Peripheral blood mononuclear cells (PBMC) were enriched by density gradient centrifugation. CD4+ and CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. Monocytes were differentiated into dendritic cells (MDDC).
  • Library clones were screened in replicates using 5,000 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 100:1. After 24 hours incubation, assay supernatants were harvested and stored at −80° C. Supernatant cytokines were analyzed using a Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
    • Data Analysis
  • Clones that induced mean IFNγ responses that were statistically different from background (Wilcoxon Rank Sum, p<0.05) and exceeded 3 standard deviations (SD) of the mean of the negative control GFP clones (N=10) were considered antigens.
  • FIG. 1 shows representative results for a single melanoma patient. Clones that induced mean IFNγ responses that were statistically different from background (Wilcoxon Rank Sum, p<0.05) and exceeded 3 standard deviations (SD) of the mean of the negative control GFP clones (N=10) were considered antigens (indicated by horizontal dotted line). CEF=positive control peptide pool. GFP=green fluorescent protein. Each symbol represents an individual measurement, horizontal line=mean. Un022 & Un023 were not included in the CD8+ library.
    • Example 2. Cohort-Specific T Cell Responses to TAAs Associated with Protective Immunity in Melanoma Patients after Checkpoint Blockade Therapy
  • Dozens of subjects were recruited into the study and cohorted based upon their clinical outcome after checkpoint inhibitor therapy. Subjects who had stable disease or tumor regression were considered protected; those who had worsening disease (tumor growth) were considered not protected. Clinical determinations were made by tumor imaging scans.
  • Briefly, blood samples were collected from 32 consented melanoma patients who had previously undergone checkpoint inhibitor therapy (one subject had two separate collections). Peripheral blood mononuclear cells (PBMC) were enriched by density gradient centrifugation. CD4+ and CD8+ T cells were sorted and non-specifically expanded using anti-CD3 and anti-CD28-coated microbeads, and CD14+ monocytes were differentiated into dendritic cells (MDDC). Library clones comprising known TAAs (labelled as Un001-023, as shown above in Table 3) were screened in duplicate using 5,000 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 100:1; ten replicates of E. coli expressing GFP were included as negative controls. Assay supernatants were harvested at 24 hours and stored at −80° C. Supernatant cytokines were analyzed using Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
    • Data Analysis
  • Clones that induced mean cytokine responses that were statistically different from background (Wilcoxon Rank Sum, p<0.05) and exceeded 3 standard deviations (SD) of the mean responses to the negative control GFP clones (N=10) were considered antigens. The mean number of antigens to which each cohort responded with each cytokine were compared to determine if differences existed between protected (Responder) and non-protected (Non-responder) cohort.
  • FIG. 2 shows cohort data for the CD4+ T cell subset. Subjects were cohorted into “Responder” (gray bars) or “Non-Responder” (black bars) groups based on clinical evaluation of disease. Using a cutoff of 3 SD above the mean of the negative control response per patient for each cytokine evaluated, the number of TAAs to which each subject responded with their CD4+ T cell subset is represented. In contrast to the Responder cohort, the Non-Responder group had minimal discernable CD4+ T cell responses, by the majority of cytokines evaluated, to any of the TAAs included in the library. Data are shown as the mean number (±SE) of TAAs to which each cohort responded with each cytokine measured.
  • FIG. 3 shows cohort data for the CD8+ T cell subset. Subjects were cohorted into “Responder” (gray bars) or “Non-Responder” (black bars) groups based on clinical evaluation of disease. Using a cutoff of 3 SD above the mean of the negative control response per patient for each cytokine evaluated, the number of TAAs to which each subject responded with their CD8+ T cell subset is represented. CD8+ T cells secreting IFNγ were undetectable in Non-Responders, but Responders had responses to a mean of ˜two TAAs. Data are shown as the mean number (±SE) of TAAs to which each cohort responded with each cytokine measured.
    • Example 3. Immune Responses to Neoantigens Identified Using ATLAS in a Non-Small Cell Lung Cancer (NSCLC) Patient Generation of the ATLAS Neoantigen Library
  • ATLAS (Genocea Biosciences) was applied to screen the entire complement of mutations identified in the tumor of a consented NSCLC patient who was successfully treated with pembrolizumab (αPD-1 antibody (Ab), every other week starting on day 0). An ATLAS library was built that expressed 201 of 202 mutations unique to this patient. Each clone contained 113 amino acids with the mutation positioned near the center of the construct and sequence-verified. Each clone was recombinantly expressed in E. coli. Protein expression was verified using a surrogate T cell assay (the B3Z hybridoma) which recognizes the C57BL/6 mouse T cell epitope SIINFEKL (SEQ ID NO: 452), which is inserted at the C-terminus of each open reading frame, upstream of the stop codon. Proteins that induced B3Z responses that exceeded 5% of the positive control (the minimal SIINFEKL (SEQ ID NO: 452) epitope pulsed onto antigen presenting cells) were considered expressed.
    • ATLAS Library Screening
  • Peripheral blood samples were collected from the NSCLC patient before and after checkpoint blockade therapy. Peripheral blood mononuclear cells (PBMC) were enriched by density gradient centrifugation. CD4+ and CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. Monocytes were differentiated into dendritic cells (MDDC).
  • CD4+ and CD8+ T cells from Day 0 and Day 42 (after 3rd injection) of treatment were screened, respectively, against 195 and 201 of the 201 library clones, as well as against 20 negative control clones expressing Neon Green (NG). Library clones were screened in duplicate using 2,000 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 250:1. After 24h incubation, assay supernatants were harvested and stored at −80° C. Supernatant cytokines were analyzed using a Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
    • Data Analysis
  • Clones that induced mean cytokine responses that were statistically different from background (Wilcoxon Rank Sum, p<0.05) and exceeded 3 standard deviations (SD) of the mean responses to the negative control Neon Green clones (N=20) were considered antigens (indicated by horizontal dotted line).
  • FIG. 4 shows good alignment between duplicate measurements of the cytokines IFNγ and TNFα for CD8+ T cell response, with over 74% of replicates falling within 1.5-fold of one another.
  • FIG. 5 shows results for IFNγ and TNFα for CD8+ T cells pre- and post-treatment (left and right panels respectively). In this NSCLC patient, 5% of mutations screened (9 of 201) were identified as neoantigens recognized by his/her peripheral blood CD8+ T cells taken pre- and post-treatment. Only 1% of the identified neoantigens were found both pre- and post-treatment. Points above the top dotted line indicate neoantigens that stimulate CD8+ T cell responses. Points below the lower dotted line indicate neoantigens that suppress and/or inhibit CD8+ T cell responses.
  • FIG. 6 shows results for IFNγ and TNFα for CD4+ T cells pre- and post-treatment (left and right panels respectively). In this NSCLC patient, 10% of mutations screened (20 of 195) were identified as neoantigens recognized by his/her peripheral blood CD4+ T cells taken pre-treatment, increasing to 17% of mutations screened (33 of 195) post-treatment. Five percent of the identified neoantigens were found both pre- and post-treatment. Points above the top dotted line indicate neoantigens that stimulate CD4+ T cell responses. Points below the lower dotted line indicate neoantigens that suppress and/or inhibit CD4+ T cell responses. These results show increased breadth of CD4+ T cell responses to neoantigens following checkpoint inhibitor therapy, particularly with respect to IFNγ.
  • TABLE 4
    summarizes results shown in FIGS. 5 and 6.
    T cell responses Pre-treatment Post-treatment Both
    CD8 +  5%  5% 1%
    CD4 + 10% 17% 5%
  • FIG. 7 shows the limited overlap between CD8+-specific T cell neoantigens identified by ATLAS and epitope prediction algorithms. MEW class I epitopes were predicted for all screened neoantigens from the NSCLC patient using three commonly used algorithms: NetMHC, NetCTLpan and IEDB, and using patient-specific haplotypes HLA-A*02:01/*32:01, HLA-B*40:01:02/*45:01:01, HLAC*06:02/*03:041 (see Rizvi et al., (2015) Science. 348(6230): 124-8). Eight of the antigens identified by ATLAS were not predicted by any of NetMHC, NetCTLpan, or IEDB. (Note that MEW class II epitopes cannot be effectively predicted using currently available algorithms.)
  • FIG. 8 further shows that epitope predictions have a high false positive rate, miss relevant stimulatory neoantigens, and are not able to identify suppressive and/or inhibitory neoantigens. Of the 137 neoantigens predicted by at least one algorithm, only 15 (or 11%) were confirmed by ATLAS to effect a CD8+ T cell response in the NSCLC patient. Six of these 15 neoantigens were found to be suppressive and/or inhibitory. Altogether, ATLAS identified 9+8 stimulatory neoantigens, and 6+3 suppressive and/or inhibitory neoantigens. Thus, 47% of stimulatory antigens found by ATLAS were missed by algorithms, and 33% of suppressive and/or inhibitory neoantigens found by ATLAS failed to be identified by algorithms.
    • Example 4. Immune Responses to the ATLAS Colorectal Cancer (CRC) Tumor Associated Antigen (TAA) Library—Single Patient Response Generation of the ATLAS Colorectal Cancer TAA Library
  • Twenty-six TAA genes (representing 23 unique genes; labelled as “taa1-26” and shown below in Table 5) were cloned into the ATLAS expression vector (Genocea Biosciences), and sequence-verified. Each TAA was recombinantly expressed in E. coli. Protein expression was verified using a surrogate T cell assay (the B3Z hybridoma) which recognizes the C57BL/6 mouse T cell epitope SIINFEKL (SEQ ID NO: 452), which is inserted at the C-terminus of each open reading frame, upstream of the stop codon. Proteins that induced B3Z responses that exceeded 5% of the positive control (the minimal SIINFEKL (SEQ ID NO: 452) epitope pulsed onto antigen presenting cells) were considered expressed.
    • ATLAS Library Screening
  • A frozen peripheral blood mononuclear cell (PBMC) vial was purchased from Bioreclamation IVT. The PBMC were derived from a 50 year-old Caucasian male who had stage IV colorectal cancer. CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. Monocytes were differentiated into dendritic cells (MDDC).
  • Library clones were screened in replicates using 5,000 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 100:1. After 24h incubation, assay supernatants were harvested and stored at −80° C. Negative controls included 13 replicates of E. coli expressing neon green (NG). Supernatant cytokines were analyzed using a Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
    • Data Analysis
  • Measurements that were below the lower limit of detection for the standard curve of each cytokine were masked. Clones that induced mean IFNγ or TNFα responses that exceeded 3 standard deviations (SD) of the mean of the negative control neon green (NG) clones (N=13) were considered antigens.
  • FIG. 9 shows representative results for a single CRC patient. Clones that induced mean IFNγ and/or TNFα responses that exceeded 3 standard deviations (SD) of the mean of the negative control NG clones (N=10) were considered antigens (indicated by horizontal dotted line, black symbols). NG=neon green. Each symbol represents an individual measurement, small horizontal line=mean of duplicate measurements. TAA coding conventions are shown in Table 5 below.
    • Example 5. T Cell Responses to CRC TAAs in a Cohort of CRC Patients
  • PBMC from 21 CRC patients were screened against a library of 26 known TAAs (shown in Table 5). CD4+ and CD8+ T cells were sorted and non-specifically expanded using anti-CD3 and anti-CD28-coated microbeads, and CD14+ monocytes were differentiated into dendritic cells (MDDC). Library clones were screened in duplicate using 5,000 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 100:1; 13 replicates of E. coli expressing neon green (NG) were included as negative controls. Assay supernatants were harvested at 24 hours and stored at −80° C. Supernatant cytokines were analyzed using Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
    • Data Analysis
  • Clones that induced mean cytokine responses that exceeded 3 standard deviations (SD) of the mean responses to the negative control NG clones (N=10) were considered antigens.
  • FIG. 10 shows data for both the CD4+ (grey bars) and CD8+ (black bars) T cell subsets. The percentage of subjects who responded to each TAA, as measured by IFNγ secretion that exceeded three standard deviations of the mean negative control (NG) response, is represented. Overall, nine of 26 antigens induced a CD8+ T cell response from at least one of the CRC patients. Three TAAs (HPSE1, HPSE2, SMAD4) were antigens for CD8+ T cells from nearly all subjects screened; two TAAs (HPSE1, HPSE2) were also antigens for each subject's CD4+ T cell subset. Results are summarized in Table 5 below.
  • TABLE 5
    Summary of T cell response rates to TAAs in the
    ATLAS colorectal cancer library
    Code TAA CD4 CD8
    taa1 BIRC5
    0 0
    taa2 CDH3 0 0
    taa3 CEACAM3 0 0
    taa4 CEACAM5 0 0
    taa5 CGB_5 0 0
    taa6 COA1 0 0
    taa7 EBAG9 0 0
    taa8 EGFR 0 0
    taa9 ELK4 0 0
    taa10 ERBB2 0 0
    taa11 EpCAM 0   8%
    taa12 HPSE1 100%  92%
    taa13 HPSE2 100%  77%
    taa14 KRAS_isoform1
    0  31%
    taa15 KRAS_isoform2
    0 0
    taa16 MAGEA3 0 0
    taa17 MUC1 0 0
    taa18 SMAD4 0 100%
    taa19 TERT.2 0  31%
    taa20 TERT.3 0  31%
    taa21 TGFBR2
    0   8%
    taa22 EBAG9_isoform1
    0 0
    taa23 TP53 0  15%
    taa24 CGB_3
    0 0
    taa25 IMPDH2 0 0
    taa26 LCK 0 0
    • Example 6. Immune Responses to Neoantigens Identified Using ATLAS in a Colorectal Cancer (CRC) Patient Generation of the ATLAS Neoantigen Library
  • ATLAS was applied to screen the entire complement of mutations identified in the tumor of a consented colorectal cancer patient. An ATLAS library was built that expressed 31 mutations unique to this patient. Each clone contained 113 amino acids with the mutation positioned near the center of the construct and sequence-verified. Each clone was recombinantly expressed in E. coli and protein expression was verified using Western Blot.
    • ATLAS Library Screening
  • Frozen peripheral blood mononuclear cells (PBMC) were purchased from Conversant Bio. After thaw, CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDC).
  • CD8+ T cells were screened against the 31 library clones, as well as against 2 negative control clones expressing Neon Green (NG). Library clones were screened using 1,500 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at −80° C. Supernatant cytokines were analyzed using a Meso Scale Discovery custom plate.
    • Data Analysis
  • Clones that induced median cytokine responses that exceeded 3 median absolute deviations (MAD) of the median responses to the negative control Neon Green clones (N=2) (indicated by horizontal dotted line in FIG. 11) were considered antigens. Clones that reduced median cytokine responses to 3 MAD below the median negative control responses were considered inhibitory and/or suppressive antigens.
  • FIG. 11 shows results for IFNγ and TNFα from the patient's CD8+ T cells. The X indicates the median response to the negative controls. Points above the top dotted line indicate neoantigens that stimulate CD8+ T cell responses (black circles). Points below the lower dotted line indicate neoantigens that inhibit and/or suppress CD8+ T cell responses (black squares). In this patient, 16% of mutations screened (5 of 31) were identified as neoantigens recognized by his/her peripheral blood CD8+ T cells. Additionally, 13% (4 of 31) of mutations screened were identified as inhibitory and/or suppressive neoantigens. There was no overlap of the neoantigens that induced IFNγ compared with TNFα, but two of the inhibitory neoantigens suppressed both IFNγ and TNFα.
  • FIGS. 12A and 12B show Venn diagrams representing the limited overlap between CD8+-specific T cell neoantigens identified by ATLAS and epitope prediction algorithms. MHC class I epitopes were predicted for all screened neoantigens using three commonly used algorithms: NetMHC, NetCTLpan and IEDB, and using patient-specific haplotypes HLA-A*30:02/*32:01, B*18:01/*14:01, C*05:01/*08:02. FIG. 12A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ≤1 for the mutant peptide but not for wild-type. FIG. 12B represents epitopes predicted to have binding affinity below 500 nM or an IEDB percentile rank of ≤1, irrespective of the wild-type counterpart predictions. In the former case, none of the neoantigens that were identified by ATLAS were predicted by algorithms, and there were six epitopes predicted that were not identified empirically (100% false positive and 100% false negative rate). For the latter, there was one neoantigen that was identified using ATLAS that was also predicted by all three algorithms used. The remaining four neoantigens were not predicted by any algorithm. There were 26 epitopes predicted that could not be confirmed by ATLAS (therefore the algorithms had a 96% false positive rate and an 80% false negative rate).
  • FIGS. 13A and 13B show Venn diagrams representing the limited overlap between CD8+-specific T cell inhibitory and/or suppressive neoantigens identified by ATLAS and epitope prediction algorithms. Epitope predictions do not discriminate between stimulatory or inhibitory and/or suppressive antigens, therefore the same MHC predictions used for FIGS. 12A and 12B were applied for the inhibitory and/or suppressive, rather than stimulatory neoantigens. FIG. 13A represents epitopes predicted that had binding affinity projected to be below 500 nM for the mutant peptide (neoantigen) but not for its wild-type counterpart, and an IEDB percentile rank of ≤1 for the mutant peptide but not for wild-type. FIG. 13B represents epitopes predicted to have binding affinity below 500 nM or an IEDB percentile rank of ≤1, irrespective of the wild-type counterpart predictions. In the former case, none of the inhibitory and/or suppressive neoantigens that were identified by ATLAS were predicted by algorithms, and there were six epitopes predicted that were not identified empirically (100% false positive and 100% false negative rate). For the latter, there was one neoantigen that was identified using ATLAS that was also predicted by one of the three algorithms (netMHCpan_MT). The remaining three neoantigens identified empirically with ATLAS were not predicted by any algorithm. Once again, there were 26 epitopes predicted that could not be confirmed by ATLAS.
    • Example 7. T Cell Response Profiling in Colorectal Carcinoma Patients Reveals an Enrichment in Responses to Specific Tumor-Associated Antigens
  • Generation of an ATLAS Tumor Associated Antigen library
  • ATLAS™ was applied to profile T cell recall responses to a set of Tumor Associated Antigens (TAAs) in 34 subjects with various stages of CRC and pre-malignant lesions in an HLA-independent manner. Twenty-six TAA genes (representing 23 unique genes, shown in Table 5) were cloned into the ATLAS expression vector and sequence verified. Each TAA was recombinantly expressed in E. coli, with expression verified using Western Blot analysis.
  • TABLE 6
    ATLAS colorectal cancer TAA library
    Antigen Name Alias long name OMIM GeneID
    CDH3 CDHP, HJMD, PCAD cadherin 3 114021 1001
    CEACAM3 CEA; CGM1; W264; carcinoembryonic antigen- 609142 1084
    W282; CD66D related cell adhesion
    molecule 3
    CEACAM5 CEA; CD66e carcinoembryonic antigen- 114890 1048
    related cell adhesion
    molecule 5
    CGB_3 CGB, CGB5, CGB7, chorionic gonadotropin 118860 1082
    CGB8, hCGB beta subunit 3
    CGB_5 CGB, HCG, hCGB chorionic gonadotropin 608825 93659
    beta subunit 5
    COA1 C7orf44, MITRAC15 cytochrome c oxidase 614769 55744
    assembly factor 1 homolog
    EBAG9 EB9, PDAF estrogen receptor binding 605772 9166
    site associated, antigen, 9
    EGFR ERBB; HER1; mENA; epidermal growth factor 131550 1956
    ERBB1; PIG61; NISBD2 receptor
    ELK4 SAP1 ETS transcription factor 600246 2005
    EpCAM ESA; KSA; M4S1; MK- epithelial cell adhesion 185535 4072
    1; DIAR5; EGP-2; molecule precursor
    EGP40; KS1/4; MIC18;
    TROP1; EGP314;
    HNPCC8; TACSTD1
    ERBB2 NEU; NGL; HER2; receptor tyrosine-protein 164870 2064
    TKR1; CD340; HER-2; kinase erbB-2
    MLN 19; HER-2/neu
    HPSE1 heparanase isoform 1 604724 10855
    HPSE2 heparanase isoform 2
    IMPDH2 IMPD2, IMPDH-II inosine monophosphate 146691 3615
    dehydrogenase 2
    KRAS C-K-RAS, CFC2, K- KRAS proto-oncogene, 190070 3845
    RAS2A, K-RAS2B, K- GTPase
    RAS4A, K-RAS4B, K-
    Ras, K
    LCK LSK; YT16; IMD22; tyrosine-protein kinase 153390 3932
    p56lck; pp58lck Lck
    MAGEA3 HIP8; HYPD; CT1.3; MAGE family member A3 300174 4102
    MAGE3; MAGEA6,
    MAGE-A3 (G-2544)
    MUC1 EMA; MCD; PEM; mucin-1 isoform 14 158340 4582
    PUM; KL-6; MAM6; precursor
    MCKD; PEMT; CD227;
    H23AG; MCKD1; MUC-
    1; ADMCKD;
    ADMCKD1; CA 15-3;
    MUC-1/X; MUC1/ZD;
    MUC-1/SEC
    SMAD4 DPC4, JIP, MADH4, SMAD family member 4 600993 4089
    MYHRS
    BIRC5 API4; EPR-1; survivin, survivin 603352 332
    BIRC5
    TERT TP2; TRT; CMM9; telomerase reverse 187270 7015
    EST2; TCS1; hTRT; transcriptase
    DKCA2; DKCB4;
    hEST2; PFBMFT1
    TGFBR2 AAT3, FAA3, LDS1B, transforming growth factor 190182 7048
    LDS2, LDS2B, MFS2, beta receptor 2
    RTIC, TAAD2, TGFR-2,
    TP53 P53; BCC7; LFS1; cellular tumor antigen p53 191170 7157
    TRP53
    OMIM = Online Mendelian Inheritance in Man database
    GeneID = NCBI database
    • ATLAS Library Screening
  • Frozen peripheral blood mononuclear cells (PBMC) were purchased from Conversant Bio (Alabama) or obtained from a collaborator at Mayo Clinic. After thaw, CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDCs).
  • Frozen peripheral blood mononuclear cells (PBMC) were purchased from Conversant Bio (Alabama) or obtained from a collaborator at Mayo Clinic. After thaw, CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDCs).
  • CD4+ and CD8+ T cells were screened against the 26 library clones, as well as against 10 negative control clones expressing Neon Green (NG). Library clones were screened using 1,000-5,000 MDDCs and 80,000 T cells, at an E. coli:MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at −80° C. Supernatant cytokines levels were analyzed using a Meso Scale Discovery custom plate.
    • Data Analysis
  • Clones that induced median cytokine responses that exceeded 2 median absolute deviations (MAD) of the median responses to the negative control Neon Green (NG) clones (N=10) (indicated by a vertical dotted line in FIG. 14 and a horizontal dotted line in FIG. 17) were considered antigens. Clones that reduced median cytokine responses to 2 MAD below the median negative control responses were considered inhibitory and/or suppressive antigens. In CRC patients, the breadth of recall responses to the 26 tested TAAs varied, but there was a strong enrichment of CD4+ and CD8+ T cell responses to a subset of 3 TAAs, which was absent in healthy individuals.
  • FIG. 14 shows response profiles to 25 CRC-associated TAAs across CRC patients. CD4+ and CD8+ T cells from CRC patients across all stages of disease were profiled for responses to 25 TAAs, using TNF-α and IFN-γ secretion as an indicator for a recall response to a putative antigen. Distributions of normalized cytokine concentrations released in response to each antigen are shown, each row represents one antigen. Dashed vertical lines indicate 2 MADs from median cytokine release in response to the NG negative control antigen. Positive values, indicated by a shift toward the right side of the plot, indicate stimulatory T cell recall responses. Negative values, indicated by a shift toward the left side of the plot, indicate inhibitory and/or suppressive T cell recall responses.
  • FIGS. 15A and 15B shows the high frequency of T cell responses to three TAAs not previously identified by algorithm. Response rates in individuals with CRC to three ATLAS-identified TAAs in comparison to three TAAs that are or were in clinical development as a therapeutic vaccine. FIG. 15A shows response rate of CD4+ and CD8+ T cells for HPSE1 and HPSE2, in comparison to MUC1, MAGEA3, and TP53. FIG. 15B shows response rate of CD8+ T cells for HPSE1, HPSE2 and SMAD4, in comparison to MUC1, MAGEA3, and TP53. Stimulatory (top panels) and inhibitory and/or suppressive (bottom panels) T cell recall responses are shown.
  • FIG. 16 shows T cell responses to selected TAAs in CRC patients with early or late stage disease (NR, no responders). Stimulatory response rates to four selected TAAs are shown for both CD4+ and CD8+ T cell subsets and TNF-α and IFN-γ cytokine release (Panel A=HPSE1; Panel B=HPSE2; Panel C=TP53; Panel D=MAGEA3). Patients were grouped by stage of disease with early stage representing stages I and II, i.e., locoregional disease, and late stage representing stages III and IV, i.e., with metastasis to lymph nodes or distant sites. There was no significant difference between response rates in early and late disease for either stimulatory responses (shown) or inhibitory and/or suppressive responses (not shown). Stage of cancer did not impact the T cell response signature.
  • FIG. 17 shows T cell responses to selected TAAs in healthy individuals and donors with various disease states. Normalized cytokine concentrations released in response to the four selected TAAs in the three cohorts are shown for CD4+ and CD8+ T cell subsets and for TNF-α and IFN-γ release (Panel A=HPSE1; Panel B=HPSE2; Panel C=TP53; Panel D=MAGEA3). Each data point represents one individual. IFN-γ release in different cohorts was compared using a Wilcoxon rank sum test. Asterisks indicate statistical significance in comparison to cytokine release in healthy donors unless otherwise indicated. * p<0.05; ** p<0.01; *** p<0.001. Significant differences based on TNF-α levels were detected across the same groups (not shown). Importantly, T cell responses to a subset of TAAs (HPSE1, HPSE2, SMAD4) in individuals with pre-malignant adenomatous polyps were similar to those in CRC patients and clearly distinguishable from the rare responses in healthy individuals. This pattern was not observed for responses to TAAs currently or previously investigated as therapeutic vaccines (MUC1, TP53, MAGEA3).
    • Example 8. Profiling of T Cell Responses to Tumor-Associated Antigens in Lung Cancer Patients Treated with Checkpoint Inhibitors Generation of an ATLAS Tumor Associated Antigen Library
  • ATLAS was applied to characterize and profile T cell responses to Tumor Associated Antigens (TAAs) in a diverse sample of lung cancer patients undergoing ICI therapy. Seventy-six TAA genes (representing 74 unique genes, shown in Table 7) were cloned into the ATLAS expression vector and sequence verified. Each TAA was recombinantly expressed in E. coli, with expression verified using Western Blot analysis.
  • TABLE 7
    ATLAS lung cancer TAA library
    Gene Name Alias Long Name OMIM GeneID
    ACTN4 ACTININ-4, FSGS, actinin alpha 4 604638 81
    FSGS1
    ACVR1 ACTRIA, ACVRLK2, activin A receptor type 1 102576 90
    ALK2, FOP, SKR1,
    TSRI, ACVR1,
    ACTIVIN
    ADH1C ADH3 alcohol dehydrogenase 1C 103730 126
    (class I), gamma
    polypeptide
    ADORA2A A2aR, ADORA2, RDC8, adenosine A2a receptor 102776 135
    A2AR
    AKAP-4 AKAP82, AKAP-4, A-kinase anchoring protein 300185 8852
    AKAP82, CT99, FSC1, 4
    HI, PRKA4, hAKAP82,
    p8, AKAP4
    ARHGEF16 GEF16, NBR Rho guanine nucleotide 27237
    exchange factor 16
    BAGE BAGE1; CT2.1 B melanoma antigen 1 605167 574
    precursor
    BLNK AGM4, BASH-S, LY57, B-cell linker 604515 29760
    SLP-65, SLP65, bca,
    BLNK
    BNC1 BNC, BSN1, HsT19447 basonuclin 1 601930 646
    BPIFA1 LUNX, NASG, PLUNC, BPI fold containing family 607412 51297
    SPLUNC1, SPURT, A member 1
    bA49G10.5
    CACNB3 CAB3, CACNLB3 calcium voltage-gated 601958 784
    channel auxiliary subunit
    beta 3
    CASP3 CPP32, CPP32B, SCA-1, caspase 3 600636 836
    CASPASE-3
    CAV1 BSCL3, CGL3, LCCNS, caveolin 1 601047 857
    MSTP085, PPH3, VIP21
    CDH1 Arc-1, BCDS1, CD324, cadherin 1 192090 999
    CDHE, ECAD, LCAM,
    UVO
    COX8C COX8-3 cytochrome c oxidase 616855 341947
    subunit 8C
    CPT1A CPT1, CPT1-L, L-CPT1 carnitine 600528 1374
    palmitoyltransferase 1A
    CTAG1A CT6.1, ESO1, LAGE-2, cancer/testis antigen 1A 300657 246100
    LAGE2A, NY-ESO-1
    CTCFL CT27; BORIS; CTCF-T; transcriptional repressor 607022 140690
    HMGB1L1; dJ579F20.2 CTCFL
    CXCL13 ANGIE, ANGIE2, BCA- C-X-C motif chemokine 605149 10563
    1, BCA1, BLC, BLR1L, ligand 13
    SCYB13
    DGKH DGKeta diacylglycerol kinase eta 604071 160851
    EEF2 EEF-2, EF-2, EF2, eukaryotic translation 130610 1938
    SCA26 elongation factor 2
    EGFR ERBB; HER1; mENA; epidermal growth factor 131550 1956
    ERBB1; PIG61; receptor
    NISBD2
    E1F5A EIF-5A1, eIF5AI, EIF5A eukaryotic translation 600187 1984
    initiation factor 5A
    FN1 CIG, ED-B, FINC, FN, fibronectin 1 135600 2335
    FNZ, GFND, GFND2,
    LETS, MSF, Fibronectin
    GAGE1 CT4.1; GAGE-1 G antigen 1 300594 2543
    GAGE4 CT4.4 G antigen 12I 300597 2576
    HLA-DRB1 major histocompatibility 142857 3123
    complex, class II, DR beta
    1
    HLA-DRB5 major histocompatibility 604776 3127
    complex, class II, DR beta
    5
    HPSE1 heparanase isoform 1 604724 10855
    HPSE2 heparanase isoform 2
    HSD17B3 EDH17B3, SDR12C2 hydroxysteroid 17-beta 605573 3293
    dehydrogenase 3
    IDE INSULYSIN insulin degrading enzyme 146680 3416
    IDO1 IDO, IDO-1, INDO indoleamine 2,3- 147435 3620
    dioxygenase 1
    IGFBP5 IBP5 insulin like growth factor 146734 3488
    binding protein 5
    IGFBP7 AGM, FSTL2, IBP-7, insulin like growth factor 602867 3490
    IGFBP-7, IGFBP-7v, binding protein 7
    IGFBPRP1, MAC25,
    PSF,
    KCNK1 DPK, HOHO, K2P1, potassium two pore 601745 3775
    K2p1.1, KCNO1, TWIK- domain channel subfamily
    1, TWIK1 K member 1
    LAMP3 CD208, DC LAMP, DC- lysosomal associated 605883 27074
    LAMP, DCLAMP, membrane protein 3
    LAMP, LAMP-3,
    TSC403
    MAGEA1 CT1.1; MAGE1 MAGE family member A1 300016 4100
    MAGEA3 HIP8; HYPD; CT1.3; MAGE family member A3 300174 4102
    MAGE3; MAGEA6,
    MAGE-A3 (G-2544)
    MAGEB2 CT3.2, DAM6, MAGE- MAGE family member B2 300098 4113
    XP-2
    MAPK13 MAPK 13, MAPK-13, mitogen-activated protein 602899 5603
    PRKM13, SAPK4, kinase 13
    p38delta
    MARCO SCARA2, SR-A6 macrophage receptor with 604870 8685
    collagenous structure
    ME1 HUMNDME, MES malic enzyme 1 154250 4199
    MIIP IIP45, IGFBP-2 migration and invasion 608772 60672
    inhibitory protein
    MMP12 HME, ME, MME, MMP- matrix metallopeptidase 12 601046 4321
    12
    MMP7 MMP-7, MPSL1, matrix metallopeptidase 7 178990 4316
    PUMP-1
    MPZL1 MPZL1b, PZR, PZR1b, myelin protein zero like 1 604376 9019
    PZRa, PZRb
    MSR1 CD204, SCARA1, SR-A, macrophage scavenger 153622 4481
    SR-AI, SR-AII, SR-AIII, receptor 1
    SRA, phSR1, ph
    MUC1 EMA; MCD; PEM; mucin-1 isoform 14 158340 4582
    PUM; KL-6; MAM6; precursor
    MCKD; PEMT; CD227;
    H23AG; MCKD1;
    MUC-1; ADMCKD;
    ADMCKD1; CA 15-3;
    MUC-1/X; MUC1/ZD;
    MUC-1/SEC
    MYNN OSZF, SBBIZ1, myoneurin 606042 55892
    ZBTB31, ZNF902
    NAGK GNK, HSA242910 N-acetylglucosamine 606828 55577
    kinase
    NAPSA KAP, Kdap, NAP1, napsin A aspartic peptidase 605631 9476
    NAPA, SNAPA
    NFYC CBF-C, CBFC, nuclear transcription factor 605344 4802
    H1TF2A, HAP5, HSM, Y subunit gamma
    NF-YC
    NKRF ITBA4, NRF NFKB repressing factor 300440 55922
    PLAU ATF, BDPLT5, QPD, plasminogen activator, 191840 5328
    UPA, URK, u-PA urokinase
    ROR1 NTRKR1, dJ537F10.1 receptor tyrosine kinase 602336 4919
    like orphan receptor 1
    RUNX1 AML1, AML1-EVI-1, runt related transcription 151385 861
    AMLCR1, CBF2alpha, factor 1
    CBFA2, EVI-1,
    PEBP2aB,
    SFTPA1 COLEC4, PSAP, PSP-A, surfactant protein A1 178630 653509
    PSPA, SFTP1B, SP-A,
    SP-A1, SPA, SPA1,
    SFTPA2 COLEC5, PSAP, PSP-A, surfactant protein A2 178642 729238
    PSPA, SFTP1B, SP-2A,
    SP-A, SPA2, SPAII
    SFTPB PSP-B, SFTB3, SFTP3, surfactant protein B 178640 6439
    SMDP1, SP-B
    SFTPC BRICD6, PSP-C, SFTP2, surfactant protein C 178620 6440
    SMDP2, SP-C
    SFTPD COLEC7, PSP-D, surfactant protein D 178635 6441
    SFTP4, SP-D
    SLC2A5 GLUT-5, GLUT5, SGT1 solute carrier family 2 138230 6518
    member 5
    SPAG9 CT89, HLC-6, HLC4, sperm associated antigen 9 605430 9043
    HLC6, JIP-4, JIP4, JLP,
    PHET, PIG6
    SSX2 SSX; HD21; CT5.2; protein SSX2 300192 6757
    CT5.2A; HOM-MEL-40
    SUGT1 SGT1 SGT1 homolog, MIS12 604098 10910
    kinetochore complex
    assembly cochaperone
    SULT1C2 ST1C1, ST1C2, sulfotransferase family 1C 602385 6819
    SULT1C1, humSULTC2 member 2
    TGFBR2 AAT3, FAA3, LDS1B, transforming growth factor 190182 7048
    LDS2, LDS2B, MFS2, beta receptor 2
    RTIC, TAAD2, TGFR-2,
    TMEM52B transmembrane protein 120939
    52B
    TP53 P53; BCC7; LFS1; cellular tumor antigen p53 191170 7157
    TRP53 isoform a
    VEGF-A VPF; VEGF; MVCD1 vascular endothelial 192240 7422
    growth factor A
    XPO7 EXP7, RANBP16 exportin 7 606140 23039
    YES1 HsT441, P61-YES, Yes, YES proto-oncogene 1, 164880 7525
    c-yes Src family tyrosine kinase
    CCDC80 DRO1, SSG1, URB, coiled-coil domain 608298 151887
    okuribin containing 80
    OMIM = Online Mendelian Inheritance in Man database
    GeneID = NCBI database
    • ATLAS Library Screening
  • Blood samples were collected from 13 consenting patients undergoing ICI therapy. Frozen peripheral blood mononuclear cells (PBMC) were purchased from Bioreclamation (New York). After thaw, CD4+ and CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDCs).
  • CD4+ and CD8+ T cells were screened against the 76 library clones, as well as against 10 negative control clones expressing Neon Green (NG). Library clones were screened using 1,000-5,000 MDDCs and 80,000 T cells, at an E. coli:MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at −80° C. Supernatant cytokines levels were analyzed using a Meso Scale Discovery custom plate.
    • Data Analysis
  • Clones that induced median cytokine responses that exceeded 2 median absolute deviations (MADs) of the median responses to the negative control Neon Green clones (N=10) (indicated by a horizontal dotted line in FIG. 18 and FIG. 19) were considered antigens. Clones that reduced median cytokine responses to two MADs below the median negative control responses were considered inhibitory and/or suppressive antigens.
  • FIG. 18 shows an exemplary empirical determination of T cell responses to profiled TAAs. Exemplary data is shown for a single lung cancer patient. T cell responses were reported as natural log concentrations back-calculated from the MSD standard curve and normalized to the patient's response to a negative control protein. A stimulatory response was defined as a TAA with a median concentration greater than two MADs above the median of the negative control replicates. This threshold is shown as the upper dashed horizontal line, and stimulatory responses are shown as filled circles. An inhibitory and/or suppressive response was defined as a TAA with a median concentration greater than two MADs of the negative control replicates below the median of the negative control replicates. This threshold is shown as the lower dashed horizontal line, and inhibitory and/or suppressive responses are shown as filled triangles.
  • FIG. 19 shows frequent CD4+ T cell responses to novel TAAs compared to previously described TAAs. Across patients, IFN-γ CD4+ T cell responses to two novel TAAs (Novel TAA1=HPSE1; Novel TAA2=HPSE2) appeared to be stronger than responses to NY-ESO-1, MUC1, and MAGEA3, three TAAs that have been utilized in cancer vaccines in clinical trials for treatment of lung cancer patients. Each point represents a patient's response to that TAA, normalized to the patient's response to an irrelevant negative control protein. Stimulatory responses, those that fall above the 2×MAD cutoff indicated by the upper horizontal dotted line, are colored black. Both the median normalized concentration and the proportion of stimulatory responses to these two TAAs were higher than those of the three other TAAs. CD8+ responses to these five TAAs were more similar across patients (not shown).
  • FIG. 20 shows that lung cancer patients develop CD4+ and CD8+ T cell responses to a broad range of TAAs. Across lung cancer patients, stimulatory CD4+ and/or CD8+ T cell responses were observed in at least one individual to a clear majority of the 76 profiled TAAs. The percent of patients that developed a stimulatory T cell response to each TAA is shown separately for CD4+ (grey bars) and CD8+ (black bars) T cells. IFN-γ responses are displayed in the top two panels, and TNF-α responses are displayed in the bottom two panels. Antigens to which patients developed both a CD4+ and a CD8+ T cell response (left panels) were differentiated from antigens to which patients developed either a CD4+ or a CD8+ T cell response (right panels).
  • FIG. 21 shows that inhibitory and/or suppressive T cell responses were detected in most profiled TAAs. Inhibitory and/or suppressive T cell responses to TAAs were observed frequently across the profiled lung cancer patients. For each profiled TAA, the percent of patients that developed an inhibitory and/or suppressive T cell response, defined as a response that is two MADs lower than the response to the negative control protein, are shown for CD4+ (white bars) and CD8+ (grey bars) T cells. IFN-γ responses are displayed in the top two panels, and TNF-α responses are displayed in the bottom two panels. Antigens to which patients developed both a CD4+ and a CD8+ T cell response (left panels) were differentiated from antigens to which patients developed either a CD4+ or a CD8+ T cell response (right panels).
    • Example 9. Neoantigen Identification Using ATLAST Across Multiple Tumor Types Generation of the ATLAS Neoantigen Library
  • ATLAS was applied to characterize and profile pre-existing T cell responses to tumor specific mutations in a diverse set of cancer patients. Tumor biopsy and normal tissue samples were collected from 19 consenting patients. Whole exome and RNA sequencing of the tumor sample and whole exome sequencing of the matched normal sample identified mutations which are unique to the tumor and not present in the germline of the patient. Each somatic protein altering mutation was expressed as individual clones in the ATLAS expression vector and sequence verified. Each clone was recombinantly expressed in E. coli, with expression verified using Western Blot analysis.
    • ATLAS Library Screening
  • Blood samples were collected from 19 consenting patients and PBMCs isolated using standard procedures. Frozen peripheral blood mononuclear cells (PBMCs) were purchased from Conversant (Alabama) or obtained from collaborators. After thaw, CD4+ and CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into myeloid derived dendritic cells (MDDCs).
  • CD4+ and CD8+ T cells were screened against the individuals' specific library clones, as well as against multiple negative control clones expressing Neon Green (NG). Library clones were screened using 1,000-5,000 MDDCs and 80,000 T cells, at an E. coli:MDDC ratio of 333:1. After 24h incubation, assay supernatants were harvested and stored at −80° C. Supernatant cytokines levels were analyzed using a Meso Scale Discovery custom plate.
    • Data Analysis
  • Clones that induced median cytokine responses that exceeded 2 median absolute deviations (MADs) of the median responses to the negative control Neon Green clones (indicated by horizontal dotted line in FIG. 22) were considered stimulatory neoantigens. Clones that reduced median cytokine responses to 2 MADs below the median negative control responses were considered inhibitory and/or suppressive neoantigens.
  • FIG. 22 shows an exemplary neoantigen screen with ATLAS identifying patient-specific CD4+ and CD8+ T cell responses. For one pancreatic cancer subject, displayed are the CD4+ and CD8+ T cell responses observed in response to each candidate neoantigen. Each dot represents a technical replicate. Horizontal dotted lines indicate the cutoffs used to define stimulatory neoantigens and inhibitory and/or suppressive neoantigens at +3 and −3 Median Absolute Deviations (MADs), respectively.
  • FIGS. 23A, 23B, 23C, and 23D show that an algorithm predicting MHC Class I binding did not accurately predict CD8+ T cell responses or type of response. The diagrams compare MHC class I algorithm-based binding predictions (NetMHCpan predictions with binding affinity cutoff of <500 nM) and T cell responses observed in ATLAS across 11 initial subjects and across all 19 subjects. FIGS. 23A and 23C show the total numbers and overlap of neoantigens predicted by algorithm and observed in ATLAS for the 11 initial subjects and for all 19 subjects, respectively. FIGS. 23B and 23D show the break-down of predictions by strong binding (<150 nM), weak binding (<500 nM), or non-binding (>=500 nM) for the 11 initial subjects and for all 19 subjects, respectively. There was no enrichment of either stimulatory or inhibitory and/or suppressive responses in CD8+ T cells across binding prediction groups.
  • FIGS. 24A and 24B show that CD8+ T cell responses identified by ATLAS to candidate stimulatory neoantigens were not enriched for any mutation type. In FIG. 24A, mutation types for the 11 initial subjects were defined as missense, in-frame, or frameshift. In FIG. 24B, mutation types for all 19 subjects were defined as short variant (a combination of missense and in-frame mutations resulting in 1-2 amino acid changes relative to wild-type gene sequence) and neoORF (a combination of frameshift and loss-of-stop-codon mutations resulting in 3 or more amino acid changes relative to wild-type gene sequence). In this example, candidate inhibitory and/or suppressive neoantigens were somewhat more frequently associated with missense or short variant mutations.
  • FIGS. 25A and 25B show that lower DNA mutant allele frequency has a moderate association with CD8+ T cell response frequency (P-value=0.037). Mutant DNA allele frequency was derived from whole exome sequencing and compared to response type observed. FIG. 25A shows results for the 11 initial subjects. FIG. 25B shows results for all 19 subjects.
  • FIGS. 26A and 26B show that detection of a mutation in RNA did not predict whether the candidate stimulatory or inhibitory/suppressive antigen has a recall response in CD8+ T cells. RNA-seq was performed on the tumor material. Somatic mutations were identified via whole exome sequencing, and the RNA-seq data was interrogated for the presence or absence of mutations identified in DNA. FIG. 26A shows results for 8 of the 11 initial subjects. FIG. 26B shows results for all 19 subjects.
  • FIGS. 27A and 27B show that CD8+ T cell responses identified by ATLAS to candidate neoantigens did not correlate with gene expression. RNA-seq was performed on the tumor material; quantitative gene expression values were calculated for each gene harboring a candidate neoantigen and compared to normalized cytokine measurements. FIG. 27A shows results for 10 of the 11 initial subjects. FIG. 27B shows results for all 19 subjects.
    • Example 10. Different Cytokine Responses to Different Neoantigens Identified Using ATLAS in a Pancreatic Cancer Patient Generation of the ATLAS Neoantigen Library
  • ATLAS was applied to screen the entire complement of mutations identified in the tumor of a consented pancreatic cancer patient. An ATLAS library was built that expressed 22 mutations unique to this patient. Each clone contained 113 amino acids with the mutation positioned near the center of the construct and sequence-verified. Each clone was recombinantly expressed in E. coli and protein expression was verified using Western Blot.
  • ATLAS Library Screening
  • Frozen peripheral blood mononuclear cells (PBMC) were purchased from Conversant Bio. After thaw, CD8+ T cells were sorted using antibody-conjugated magnetic beads and non-specifically expanded with anti-CD3 and anti-CD28 stimulation. CD14+ monocytes were also sorted using antibody-conjugated magnetic beads and differentiated in vitro into dendritic cells (MDDC).
  • CD8+ T cells were screened against the 22 library clones, as well as against a negative control clones expressing Neon Green (NG). Library clones were screened using 5,000 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 333:1. After 19.5 h incubation, assay supernatants were harvested and stored at −80° C. Supernatant cytokines CM-CSF, IFNγ, IL-10, MIF, TNFα, and TRAIL were analyzed using a Meso Scale Discovery custom plate.
    • Data Analysis
  • FIG. 28 shows the different CM-CSF, IFNγ, IL-10, MIF, TNFα, and TRAIL response profiles elicited by six representative neoantigens in a screen of CD8+ T cells from the patient. Each panel corresponds to one neoantigen (denoted G-3618, G-3624, G-3620, G-3627, G-3617, and G-3632). The horizontal line in each panel indicates the median response to the negative controls. Bars above the horizontal line indicate stimulation of cytokine secretion. Bars below the horizontal line indicate inhibition and/or suppression of cytokine secretion. The panels illustrate the different cytokine responses elicited by each neoantigen.
    • Example 11. T Cell Responses to VEGF in a Cohort of Cancer Patients and Healthy Donors
  • PBMC from eight cancer patients (seven lung cancer, one colorectal cancer) and 13 healthy donors were screened in duplicate against VEGF, a known TAA. CD8+ T cells were sorted and non-specifically expanded using anti-CD3 and anti-CD28-coated microbeads, and CD14+ monocytes were differentiated into dendritic cells (MDDC). Library clones were screened in duplicate using 5,000 MDDC and 80,000 T cells, at an E. coli:MDDC ratio of 100:1; replicates of E. coli expressing neon green (NG) were included as negative controls. Assay supernatants were harvested at 24 hr and stored at −80° C. Supernatant cytokines were analyzed using Meso Scale Discovery V-PLEX Proinflammatory Panel 1 (human) Kit.
    • Data Analysis
  • Clones that induced mean cytokine responses that exceeded 2 median average deviations (MAD) of the median responses to the negative control NG clones (N=10) were considered antigens. FIG. 29 shows CD8+ T cell data for healthy donors (black bars) and cancer patients (white bars). The median log cytokine response normalized to neon green are indicated for each subject cohort. When analyzed by IFNγ secretion, there was a large inhibitory response in the healthy donor cohort, that greatly exceeded the inhibitory responses in the cancer patient cohort. Conversely, there was a greater median inhibitory response in the cancer cohort when TNFα secretion was considered.
    • Example 12. In Vitro Immunization Using Combination of 3 TAAs Protocol for In Vitro Immunization
  • PBMCs from healthy donors are enriched using standard protocols. Washed PBMCs are resuspended in supplemented RPMI-1640 medium. 100 μL cells (2×106cell/mL) are added into each well of a 96-well flat-bottom assay plate. Overlapping peptides corresponding to TAAs HPSE1, HPSE2, SMAD4, MUC1, MAGEA3, and TP53 were added to cultures at a final concentration of 50 μg/mL. Cultures are incubated for 5 days, the peptide-containing medium removed, then cultures provided with human IL-2 (10 U/mL) for 11 days, with IL-2-containing medium being replenished every 3 days. The incubation time of 5 days with peptide plus 11 days with IL-2 constitutes one cycle. Primary cultures are subsequently restimulated with the same peptides (50 ng/mL) on day 16 to begin the next cycle. Irradiated (4000 rad) autologous peripheral blood mononuclear cells (5×105) are added in a volume of 50 μL in complete medium as APCs. An ELISPOT is performed on an aliquot of cells at the end of each cycle to observe de novo responses to the peptides.
  • LISTING OF SEQUENCES
    Heparanase isoform 1, preproprotein, NP_001092010.1, NP_006656.2 (SEQ ID NO:
    6)
    1 mllrskpalp pplmllllgp lgplspgalp rpaqaqdvvd ldfftqeplh lvspsflsvt
    61 idanlatdpr flillgspkl rtlarglspa ylrfggtktd flifdpkkes tfeersywqs
    121 qvngdickyg sippdveekl rlewpyqeql llrehyqkkf knstysrssv dvlytfancs
    181 gldlifglna llrtadlqwn ssnaqllldy csskgynisw elgnepnsfl kkadifings
    241 qlgedfiqlh kllrkstfkn aklygpdvgq prrktakmlk sflkaggevi dsvtwhhyyl
    301 ngrtatkedf lnpdvldifi ssvqkvfqvv estrpgkkvw lgetssaygg gapllsdtfa
    361 agfmwldklg lsarmgievv mrqvffgagn yhlvdenfdp lpdywlsllf kklvgtkvlm
    421 asvqgskrrk lrvylhctnt dnprykegdl tlyainlhnv tkylrlpypf snkqvdkyll
    481 rplgphglls ksvqlngltl kmvddqtlpp lmekplrpgs slglpafsys ffvirnakva
    541 aci
    Heparanase isoform 2, preproprotein, NP_001159970.1 (SEQ ID NO: 7)
    1 mllrskpalp pplmllllgp lgplspgalp rpaqaqdvvd ldfftqeplh lvspsflsvt
    61 idanlatdpr flillgspkl rtlarglspa ylrfggtktd flifdpkkes tfeersywqs
    121 qvngdickyg sippdveekl rlewpyqeql llrehyqkkf knstysrssv dvlytfancs
    181 gldlifglna llrtadlqwn ssnaqllldy csskgynisw elgnepnsfl kkadifings
    241 qlgedfiqlh kllrkstfkn aklygpdvgq prrktakmlk sflkaggevi dsvtwhhyyl
    301 ngrtatkedf lnpdvldifi ssvqkvfqdy wlsllfkklv gtkvlmasvq gskrrklrvy
    361 lhctntdnpr ykegdltlya inlhnvtkyl rlpypfsnkq vdkyllrplg phgllsksvq
    421 lngltlkmvd dqtlpplmek plrpgsslgl pafsysffvi rnakvaaci
    SMAD family member 4 , mothers against decapentaplegic homolog 4, NP_005350.1
    (SEQ ID NO: 8)
    1 mdnmsitntp tsndaclsiv hslmchrqgg esetfakrai eslvkklkek kdeldslita
    61 ittngahpsk cvtiqrtldg rlqvagrkgf phviyarlwr wpdlhknelk hvkycqyafd
    121 lkcdsvcvnp yhyervvspg idlsgltlqs napssmmvkd eyvhdfegqp slsteghsiq
    181 tiqhppsnra stetystpal lapsesnats tanfpnipva stsqpasilg gshsegllqi
    241 asgpqpgqqq ngftgqpaty hhnstttwtg srtapytpnl phhqnghlqh hppmpphpgh
    301 ywpvhnelaf qppisnhpap eywcsiayfe mdvqvgetfk vpsscpivtv dgyvdpsggd
    361 rfclgqlsnv hrteaierar lhigkgvqle ckgegdvwvr clsdhavfvq syyldreagr
    421 apgdavhkiy psayikvfdl rqchrqmqqq aataqaaaaa qaaavagnip gpgsvggiap
    481 aislsaaagi gvddlrrlci lrmsfvkgwg pdyprqsike tpcwieihlh ralqlldevl
    541 htmpiadpqp ld
    Cadherin 3, isoform 1 preproprotein, NP_001784.2 (SEQ ID NO: 9)
    1 mglprgplas llllqvcwlq caaseperav freaevtlea ggaegepgqa lgkvfmgcpg
    61 qepalfstdn ddftvrnget vqerrslker nplkifpskr ilrrhkrdwv vapisvpeng
    121 kgpfpqrlnq lksnkdrdtk ifysitgpga dsppegvfav eketgwllln kpldreeiak
    181 yelfghavse ngasvedpmn isiivtdqnd hkpkftqdtf rgsvlegvlp gtsvmqvtat
    241 deddaiytyn gvvaysihsq epkdphdlmf tihrstgtis vissgldrek vpeytltiqa
    301 tdmdgdgstt tavavveild andnapmfdp qkyeahvpen avghevqrlt vtdldapnsp
    361 awratylimg gddgdhftit thpesnqgil ttrkgldfea knqhtlyvev tneapfvlkl
    421 ptstativvh vedvneapvf vppskvvevq egiptgepvc vytaedpdke nqkisyrilr
    481 dpagwlamdp dsgqvtavgt ldredeqfvr nniyevmvla mdngsppttg tgtllltlid
    541 vndhgpvpep rqiticnqsp vrqvlnitdk dlsphtspfq aqltddsdiy wtaevneegd
    601 tvvlslkkfl kqdtydvhls lsdhgnkeql tviratvcdc hghvetcpgp wkggfilpvl
    661 gavlallfll lvllllvrkk rkikeplllp eddtrdnvfy ygeegggeed qdyditqlhr
    721 glearpevvl rndvaptiip tpmyrprpan pdeignfiie nlkaantdpt appydtllvf
    781 dyegsgsdaa slssltssas dqdqdydyln ewgsrfkkla dmygggedd
    Cadherin 3, isoform 2 precursor, NP_001304124.1 (SEQ ID NO: 10)
    1 mglprgplas llllqvcwlq caasepcrav freaevtlea ggaeqepgqa lgkvfmgcpg
    61 qepalfstdn ddftvrnget vqerrslker nplkifpskr ilrrhkrdwv vapisvpeng
    121 kgpfpqrlnq lksnkdrdtk ifysitgpga dsppegvfav eketgwllln kpldreeiak
    181 yelfghavse ngasvedpmn isiivtdqnd hkpkftqdtf rgsvlegvlp gtsvmqvtat
    241 deddaiytyn gvvaysihsq epkdphdlmf tihrstgtis vissgldrek vpeytltiqa
    301 tdmdgdgstt tavavveild andnapmfdp qkyeahvpen avghevqrlt vtdldapnsp
    361 awratylimg gddgdhftit thpesnqgil ttrkgldfea knqhtlyvev tneapfvlkl
    421 ptstativvh vedvneapvf vppskvvevq egiptgepvc vytaedpdke nqkisyrilr
    481 dpagwlamdp dsgqvtavgt ldredeqfvr nniyevmvla mdngsppttg tgtllltlid
    541 vndhgpvpep rqiticnqsp vrqvlnitdk dlsphtspfq aqltddsdiy wtaevneegd
    601 tvvlslkkfl kqdtydvhls lsdhgnkeql tviratvcdc hghvetcpgp wkggfilpvl
    661 gavlallfll lvllllvrkk rkikeplllp eddtrdnvfy ygeegggeed qdyditqlhr
    721 glearpevvl rndvaptiip tpmyrprpan pdeignfiie grgergsqrg ngglqlargr
    781 trrs
    Cadherin 3, isoform 3, NP_001304125.1 (SEQ ID NO: 11)
    1 mgcpgqepal fstdnddftv rngetvgerr slkernplki fpskrilrrh krdwvvapis
    61 vpengkgpfp qrlnqlksnk drdtkifysi tgpgadsppe gvfaveketg wlllnkpldr
    121 eeiakyelfg havsengasv edpmnisiiv tdqndhkpkf tqdtfrgsvl egvlpgtsvm
    181 qvtatdedda iytyngvvay sihsqepkdp hdlmftihrs tgtisvissg ldrekvpeyt
    241 ltiqatdmdg dgstttavav veildandna pmfdpqkyea hvpenavghe vqrltvtdld
    301 apnspawrat ylimggddgd hftitthpes nqgilttrkg ldfeaknqht lyvevtneap
    361 fvlklptsta tivvhvedvn eapvfvppsk vvevqegipt gepvcvytae dpdkenqkis
    421 yrilrdpagw lamdpdsgqv tavgtldred eqfvrnniye vmvlamdngs ppttgtgtll
    481 ltlidvndhg pvpeprqiti cnqspvrqvl nitdkdlsph tspfqaqltd dsdiywtaev
    541 neegdtvvls lkkflkqdty dvhlslsdhg nkeqltvira tvcdchghve tcpgpwkggf
    601 ilpvlgavla llflllvlll lvrkkrkike plllpeddtr dnvfyygeeg ggeedqdydi
    661 tqlhrglear pevvlrndva ptiiptpmyr prpanpdeig nfiienlkaa ntdptappyd
    721 tllvfdyegs gsdaaslssl tssasdqdqd ydylnewgsr fkkladmygg gedd
    Chorionic gonadotropin beta subunit 3, precursor, NP_000728.1 (SEQ ID NO: 12)
    1 memfqgllll lllsmggtwa skeplrprcr pinatlavek egcpvcitvn tticagycpt
    61 mtrvlqgvlp alpqvvcnyr dvrfesirlp gcprgvnpvv syavalscqc alcrrsttdc
    121 ggpkdhpltc ddprfqdsss skapppslps psrlpgpsdt pilpq
    Chorionic gonadotropin beta subunit 5, precursor, NP_149032.1 (SEQ ID NO: 13)
    1 memfggllll lllsmggtwa skeplrprcr pinatlavek egcpvcitvn tticagycpt
    61 mtrvlqgvlp alpqvvcnyr dvrfesirlp gcprgvnpvv syavalscqc alcrrsttdc
    121 ggpkdhpltc ddprfqdsss skapppslps psrlpgpsdt pilpq
    Cytochrome c oxidase assembly factor 1 homolog, isoform a, NP_001308126.1,
    NP_001308127.1, NP_001308128.1, NP_001308129.1, NP_001337853.1,
    NP_001337854.1, NP_001337855.1, NP_001337856.1, NP_060694.2 (SEQ ID NO: 14)
    1 mmwqkyagsr rsmplgaril fhgvfyaggf aivyyliqkf hsralyykla veqlqshpea
    61 qealgppini hylklidren fvdivdaklk ipvsgskseg llyvhssrgg pfqrwhldev
    121 flelkdgqqi pvfklsgeng devkke
    Cytochrome c oxidase assembly factor 1 homolog, isoform b, NP_001308130.1
    (SEQ ID NO: 15)
    1 mplgarilfh gvfyaggfai vyyliqkfhs ralyyklave qlqshpeaqe algpplnihy
    61 lklidrenfv divdaklkip vsgsksegll yvhssrggpf qrwhldevfl elkdgqqipv
    121 fklsgengde vkke
    Cytochrome c oxidase assembly factor 1 homolog, isoform c, NP_001308131.1,
    NP_001308132.1, NP_001308133.1, NP_001308134.1 (SEQ ID NO: 16)
    1 mmwqkyagsr rsmplgaril fhgvfyaggf aivyyliqsk ypasrlrpdl llacscssir
    61 gnt
    Cytochrome c oxidase assembly factor 1 homolog, isoform d, NP_001337857.1
    (SEQ ID NO: 17)
    1 mqeaggqclw eqgsfstvcs mpgalplcit sfkfhsraly yklaveqlqs hpeaqealgp
    61 pinihylkli drenfvdivd aklkipvsgs ksegllyvhs srggpfqrwh ldevflelkd
    121 gqqipvfkls gengdevkke
    Estrogen receptor binding site associated, antigen, 9, NP_001265867.1,
    NP_004206.1, NP_936056.1, NP_001308129.1, (SEQ ID NO: 18)
    1 maitqfrlfk fctclatvfs flkrlicrsg rgrklsgdqi tlpttvdyss vpkqtdveew
    61 tswdedapts vkieggngnv atqqnsleql epdyfkdmtp tirktqkivi kkreplnfgi
    121 pdgstgfssr laatqdlpfi hqsselgdld twqentnawe eeedaawqae evlrqqklad
    181 rekraaeqqr kkmekeaqrl mkkeqnkigv kls
    ETS transcription factor, isoform a, NP_001964.2 (SEQ ID NO: 19)
    1 mdsaitlwqf llqllqkpqn khmicwtsnd gqfkllqaee varlwgirkn kpnmnydkls
    61 ralryyyvkn iikkvngqkf vykfvsypei lnmdpmtvgr iegdceslnf sevsssskdv
    121 enggkdkppq pgaktssrnd yihsglyssf tlnslnssnv klfklikten paeklaekks
    181 pqeptpsvik fvttpskkpp vepvaatisi gpsispssee tiqaletlvs pklpsleapt
    241 sasnvmtafa ttppissipp lqepprtpsp plsshpdidt didsvasqpm elpenlslep
    301 kdqdsvllek dkvnnssrsk kpkglelapt lvitssdpsp lgilspslpt asltpaffsq
    361 tpiiltpspl lssihfwstl spvaplspar lqgantlfqf psvlnshgpf tlsgldgpst
    421 pgpfspdlqk t
    ETS transcription factor, isoform b, NP_068567.1 (SEQ ID NO: 20)
    1 mdsaitlwqf llqllqkpqn khmicwtsnd gqfkllqaee varlwgirkn kpnmnydkls
    61 ralryyyvkn iikkvngqkf vykfvsypei lnmdpmtvgr iegdceslnf sevsssskdv
    121 enggkdkppq pgaktssrnd yihsglyssf tlnslnssnv klfklikten paeklaekks
    181 pqeptpsvik fvttpskkpp vepvaatisi gpsispssee tiqaletivs pklpsleapt
    241 sasnvmtafa ttppissipp lqepprtpsp plsshpdidt didsvasqpm elpenlslep
    301 kdqdsvllek dkvnnssrsk kpkglelapt lvitssdpsp lgilspslpt asltpaffsq
    361 vacslfmvsp llsficpfkq iqnlytqvcf lllrfvlerl cvtvm
    Receptor tyrosine-protein kinase erbB-2, isoform a precursor, NP_004439.2
    (SEQ ID NO: 21)
    1 melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly qgcqvvqgnl
    61 eltylptnas lsflgdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng
    121 dplnnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdtilwk difhknnqla
    181 ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc
    241 aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp
    301 ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan
    361 iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf etleeitgyl yisawpdslp
    421 dlsvfqnlqv irgrilhnga ysltlqglgi swlglrslre lgsglalihh nthlcfvhtv
    481 pwdqlfrnph qallhtanrp edecvgegla chqlcarghc wgpgptqcvn csqflrgqec
    541 veecrvlqgl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc
    601 psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisavvg
    661 illvvvlgvv fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel
    721 rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp
    781 yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr
    841 lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft
    901 hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm
    961 idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl dstfyrslle dddmgdlvda
    1021 eeylvpqqgf fcpdpapgag gmvhhrhrss strsgggdlt lglepseeea prsplapseg
    1081 agsdvfdgdl gmgaakglqs lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv
    1141 nqpdvrpqpp spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq
    1201 ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg ldvpv
    Receptor tyrosine-protein kinase erbB-2, isoform b, NP_001005862.1 (SEQ ID
    NO: 22)
    1 mklrlpaspe thldmlrhly qgcqvvqgnl eltylptnas lsflqdiqev qgyvliahnq
    61 vrqvplqrlr ivrgtqlfed nyalavldng dplnnttpvt gaspgglrel qlrslteilk
    121 ggvliqrnpq lcyqdtilwk difhknnqla ltlidtnrsr achpcspmck gsrcwgesse
    181 dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs dclaclhfnh sgicelhcpa
    241 lvtyntdtfe smpnpegryt fgascvtacp ynylstdvgs ctlvcplhnq evtaedgtqr
    301 cekcskpcar vcyglgmehl revravtsan iqefagckki fgslaflpes fdgdpasnta
    361 plqpeqlqvf etleeitgyl yisawpdslp dlsvfqnlqv irgrilhnga ysltlqglgi
    421 swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qallhtanrp edecvgegla
    481 chqlcarghc wgpgptqcvn csqflrgqec veecrvlqgl preyvnarhc lpchpecqpq
    541 ngsvtcfgpe adqcvacahy kdppfcvarc psgvkpdlsy mpiwkfpdee gacqpcpinc
    601 thscvdlddk gcpaeqrasp ltsiisavvg illvvvlgvv fgilikrrqq kirkytmrrl
    661 lqetelvepl tpsgampnqa qmrilketel rkvkvlgsga fgtvykgiwi pdgenvkipv
    721 aikvlrents pkankeilde ayvmagvgsp yvsrllgicl tstvqlvtql mpygclldhv
    781 renrgrlgsq dllnwcmqia kgmsyledvr lvhrdlaarn vlvkspnhvk itdfglarll
    841 dideteyhad ggkvpikwma lesilrrrft hqsdvwsygv tvwelmtfga kpydgipare
    901 ipdllekger lpqppictid vymimvkcwm idsecrprfr elvsefsrma rdpqrfvviq
    961 nedlgpaspl dstfyrslle dddmgdlvda eeylvpqqgf fcpdpapgag gmvhhrhrss
    1021 strsgggdlt lglepseeea prsplapseg agsdvfdgdl gmgaakglqs lpthdpsplq
    1081 rysedptvpl psetdgyvap ltcspqpeyv nqpdvrpqpp spregplpaa rpagatlerp
    1141 ktlspgkngv vkdvfafgga venpeyltpq ggaapqphpp pafspafdnl yywdqdpper
    1201 gappstfkgt ptaenpeylg ldvpv
    Receptor tyrosine-protein kinase erbB-2, isoform c, NP_001276865.1 (SEQ ID
    NO: 23)
    1 mprgswkpqv ctgtdmklrl paspethldm lrhlyqgcqv vqgnleltyl ptnaslsflq
    61 diqevqgyvl iahnqvrqvp lqrlrivrgt qlfednyala vldngdplnn ttpvtgaspg
    121 glrelqlrsl teilkggvli qrnpqlcyqd tilwkdifhk nnqlaltlid tnrsrachpc
    181 spmckgsrcw gessedcqsl trtvcaggca rckgplptdc cheqcaagct gpkhsdclac
    241 lhfnhsgice lhcpalvtyn tdtfesmpnp egrytfgasc vtacpynyls tdvgsctlvc
    301 plhnqevtae dgtqrcekcs kpcarvcygl gmehlrevra vtsaniqefa gckkifgsla
    361 flpesfdgdp asntaplqpe qlqvfetlee itgylyisaw pdslpdlsvf qnlqvirgri
    421 lhngaysltl gglgiswlgl rslrelgsgl alihhnthlc fvhtvpwdql frnphqallh
    481 tanrpedecv geglachqlc arghcwgpgp tqcvncsqfl rgqecveecr vlqglpreyv
    541 narhclpchp ecqpqngsvt cfgpeadqcv acahykdppf cvarcpsgvk pdlsympiwk
    601 fpdeegacqp cpincthscv dlddkgcpae qraspltsii savvgillvv vlgvvfgili
    661 krrqqkirky tmrrllqete lvepltpsga mpnqaqmril ketelrkvkv lgsgafgtvy
    721 kgiwipdgen vkipvaikvl rentspkank eildeayvma gvgspyvsrl lgicltstvq
    781 lvtqlmpygc lldhvrenrg rlgsqdllnw cmqiakgmsy ledvrlvhrd laarnvlvks
    841 pnhvkitdfg larlldidet eyhadggkvp ikwmalesil rrrfthqsdv wsygvtvwel
    901 mtfgakpydg ipareipdll ekgerlpqpp ictidvymim vkcwmidsec rprfrelvse
    961 fsrmardpqr fvviqnedlg paspldstfy rslledddmg dlvdaeeylv pqqgffcpdp
    1021 apgaggmvhh rhrssstrsg ggdltlglep seeeaprspl apsegagsdv fdgdlgmgaa
    1081 kglqslpthd psplqrysed ptvplpsetd gyvapltcsp qpeyvnqpdv rpqppspreg
    1141 plpaarpaga tlerpktlsp gkngvvkdvf afggavenpe yltpqggaap qphpppafsp
    1201 afdnlyywdq dppergapps tfkgtptaen peylgldvpv
    Receptor tyrosine-protein kinase erbB-2, isoform d precursor, NP_001276866.1
    (SEQ ID NO: 24)
    1 melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly qgcqvvqgnl
    61 eltylptnas lsflqdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng
    121 dplnnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdtilwk difhknnqla
    181 ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc
    241 aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp
    301 ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan
    361 iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf etleeitgyl yisawpdslp
    421 dlsvfqnlqv irgrilhnga ysltlqglgi swlglrslre lgsglalihh nthlcfvhtv
    481 pwdqlfrnph qallhtanrp edecvgegla chqlcarghc wgpgptqcvn csqflrggec
    541 veecrvlqgl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc
    601 psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisavvg
    661 illvvvlgvv fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel
    721 rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp
    781 yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr
    841 lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft
    901 hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm
    961 idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl dstfyrslle dddmgdlvda
    1021 eeylvpqqgf fcpdpapgag gmvhhrhrss strnm
    Receptor tyrosine-protein kinase erbB-2, isoform e, NP_001276867.1 (SEQ ID
    NO: 25)
    1 mklrlpaspe thldmlrhly qgcqvvqgnl eltylptnas lsflqdiqev qgyvliahnq
    61 vrqvplqrlr ivrgtqlfed nyalavldng dplnnttpvt gaspgglrel qlrslteilk
    121 ggvliqrnpq lcyqdtilwk difhknnqla ltlidtnrsr achpcspmck gsrcwgesse
    181 dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs dclaclhfnh sgicelhcpa
    241 lvtyntdtfe smpnpegryt fgascvtacp ynylstdvgs ctlvcplhnq evtaedgtqr
    301 cekcskpcar vcyglgmehl revravtsan iqefagckki fgslaflpes fdgdpasnta
    361 plqpeqlqvf etleeitgyl yisawpdslp dlsvfqnlqv irgrilhnga ysltlqglgi
    421 swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qallhtanrp edecvgegla
    481 chqlcarghc wgpgptqcvn csqflrgqec veecrvlqgl preyvnarhc lpchpecqpq
    541 ngsvtcfgpe adqcvacahy kdppfcvarc psgvkpdlsy mpiwkfpdee gacqpcpinc
    601 ths
    Inosine monophosphate dehydrogenase 2 , NP_000875.2 (SEQ ID NO: 26)
    1 madylisggt syvpddglta qqlfncgdgl tyndflilpg yidftadqvd ltsaltkkit
    61 lktplvsspm dtvteagmai amaltggigf ihhnctpefq anevrkvkky eqgfitdpvv
    121 lspkdrvrdv feakarhgfc gipitdtgrm gsrlvgiiss rdidflkeee hdcfleeimt
    181 kredlvvapa gitlkeanei lqrskkgklp ivneddelva iiartdlkkn rdyplaskda
    241 kkqllcgaai gtheddkyrl dllaqagvdv vvldssqgns ifqinmikyi kdkypnlqvi
    301 ggnvvtaaqa knlidagvda lrvgmgsgsi citqevlacg rpqatavykv seyarrfgvp
    361 viadggiqnv ghiakalalg astvmmgsll aatteapgey ffsdgirlkk yrgmgsldam
    421 dkhlssqnry fseadkikva qgvsgavqdk gsihkfvpyl iagiqhscqd igaksltqvr
    481 ammysgelkf ekrtssaqve ggvhslhsye krlf
    KRAS proto-oncogene, GTPase, isoform a, NP_203524.1 (SEQ ID NO: 27)
    1 mteyklvvvg aggvgksalt iqliqnhfvd eydptiedsy rkqvvidget clldildtag
    61 qeeysamrdq ymrtgegflc vfainntksf edihhyreqi krvkdsedvp mvlvgnkcdl
    121 psrtvdtkqa qdlarsygip fietsaktrq rvedafytlv reirqyrlkk iskeektpgc
    181 vkikkciim
    KRAS proto-oncogene, GTPase, isoform b, NP_004976.2 (SEQ ID NO: 28)
    1 mteyklvvvg aggvgksalt iqliqnhfvd eydptiedsy rkqvvidget clldildtag
    61 qeeysamrdq ymrtgegflc vfainntksf edihhyreqi krvkdsedvp mvlvgnkcdl
    121 psrtvdtkqa qdlarsygip fietsaktrq gvddafytlv reirkhkekm skdgkkkkkk
    181 sktkcvim
    Transforming growth factor beta receptor 2, isoform A precursor,
    NP_001020018.1 (SEQ ID NO: 29)
    1 mgrgllrglw plhivlwtri astipphvqk sdvemeaqkd eiicpscnrt ahplrhinnd
    61 mivtdnngav kfpqlckfcd vrfstcdnqk scmsncsits icekpqevcv avwrkndeni
    121 tletvchdpk lpyhdfiled aaspkcimke kkkpgetffm cscssdecnd niifseeynt
    181 snpdlllvif qvtgisllpp lgvaisviii fycyrvnrqq klsstwetgk trklmefseh
    241 caiileddrs disstcanni nhntellpie ldtlvgkgrf aevykaklkq ntseqfetva
    301 vkifpyeeya swktekdifs dinlkhenil qfltaeerkt elgkqywlit afhakgnlqe
    361 yltrhviswe dlrklgssla rgiahlhsdh tpcgrpkmpi vhrdlkssni lvkndltccl
    421 cdfglslrld ptlsvddlan sgqvgtarym apevlesrmn lenvesfkqt dvysmalvlw
    481 emtsrcnavg evkdyeppfg skvrehpcve smkdnvlrdr grpeipsfwl nhqgiqmvce
    541 tltecwdhdp earltaqcva erfselehld rlsgrscsee kipedgslnt tk
    Transforming growth factor beta receptor 2, isoform B precursor, NP_003233.4
    (SEQ ID NO: 30)
    1 mgrgllrglw plhivlwtri astipphvqk svnndmivtd nngavkfpql ckfcdvrfst
    61 cdnqkscmsn csitsicekp qevcvavwrk ndenitletv chdpklpyhd filedaaspk
    121 cimkekkkpg etffmcscss decndniifs eeyntsnpdl llvifqvtgi sllpplgvai
    181 sviiifycyr vnrqqklsst wetgktrklm efsehcaiil eddrsdisst canninhnte
    241 llpieldtlv gkgrfaevyk aklkqntseq fetvavkifp yeeyaswkte kdifsdinlk
    301 henilqflta eerktelgkq ywlitafhak gnlqeyltrh viswedlrkl gsslargiah
    361 lhsdhtpcgr pkmpivhrdl kssnilvknd ltcclcdfgl slrldptlsv ddlansgqvg
    421 tarymapevl esrmnlenve sfkqtdvysm alvlwemtsr cnavgevkdy eppfgskvre
    481 hpcvesmkdn vlrdrgrpei psfwlnhqgi qmvcetltec wdhdpearlt aqcvaerfse
    541 lehldrlsgr scseekiped gslnttk
    Actinin alpha 4, isoform 1, NP_004915.2 (SEQ ID NO: 31)
    1 mvdyhaanqs yqygpssagn gaggggsmgd ymaqeddwdr dllldpawek qqrktftawc
    61 nshlrkagtq ienidedfrd glklmlllev isgerlpkpe rgkmrvhkin nvnkaldfia
    121 skgvklvsig aeeivdgnak mtlgmiwtii lrfaiqdisv eetsakegll lwcqrktapy
    181 knvnvqnfhi swkdglafna lihrhrpeli eydklrkddp vtnlnnafev aekyldipkm
    241 ldaedivnta rpdekaimty vssfyhafsg aqkaetaanr ickvlavnqe nehlmedyek
    301 lasdllewir rtipwledrv pqktiqemqg kledfrdyrr vhkppkvqek cqleinfntl
    361 qtklrlsnrp afmpsegkmv sdinngwqhl eqaekgyeew llneirrler ldhlaekfrq
    421 kasiheawtd gkeamlkhrd yetatlsdik alirkheafe sdlaahqdrv eqiaaiaqel
    481 neldyydshn vntrcqkicd qwdalgslth srrealekte kqleaidqlh leyakraapf
    541 nnwmesamed lqdmfivhti eeieglisah dqfkstlpda drereailai hkeaqriaes
    601 nhiklsgsnp yttvtpqiin skwekvqqlv pkrdhallee qskqqsnehl rrqfasqanv
    661 vgpwiqtkme eigrisiemn gtledqlshl kqyersivdy kpnldlleqq hqliqealif
    721 dnkhtnytme hirvgweqll ttiartinev enqiltrdak gisqeqmqef rasfnhfdkd
    781 hggalgpeef kaclislgyd vendrqgeae fnrimslvdp nhsglvtfqa fidfmsrett
    841 dtdtadqvia sfkvlagdkn fitaeelrre lppdqaeyci armapyqgpd avpgaldyks
    901 fstalygesd l
    Actinin alpha 4, isoform 2, NP_001308962.1 (SEQ ID NO: 32)
    1 mvdyhaanqs yqygpssagn gaggggsmgd ymaqeddwdr dllldpawek qqrktftawc
    61 nshlrkagtq ienidedfrd glklmlllev isgerlpkpe rgkmrvhkin nvnkaldfia
    121 skgvklvsig aeeivdgnak mtlgmiwtii lrfaiqdisv eetsakegll lwcqrktapy
    181 knvnvqnfhi swkdglafna lihrhrpeli eydklrkddp vtnlnnafev aekyldipkm
    241 ldaedivgtl rpdekaimty vscfyhafsg aqkaetaanr ickvlavnqe nehlmedyek
    301 lasdllewir rtipwledrv pqktiqemqq kledfrdyrr vhkppkvqek cqleinfntl
    361 qtklrlsnrp afmpsegkmv sdinngwqhl eqaekgyeew llneirrler ldhlaekfrq
    421 kasiheawtd gkeamlkhrd yetatlsdik alirkheafe sdlaahqdrv eqiaaiaqel
    481 neldyydshn vntrcqkicd qwdalgslth srrealekte kqleaidqlh leyakraapf
    541 nnwmesamed lqdmfivhti eeieglisah dqfkstlpda drereailai hkeaqriaes
    601 nhiklsgsnp yttvtpqiin skwekvqqlv pkrdhallee qskqqsnehl rrqfasqanv
    661 vgpwiqtkme eigrisiemn gtledqlshl kqyersivdy kpnldlleqq hqliqealif
    721 dnkhtnytme hirvgweqll ttiartinev enqiltrdak gisqeqmqef rasfnhfdkk
    781 qtgsmdsddf rallistgys lgeaefnrim slvdpnhsgl vtfqafidfm srettdtdta
    841 dqviasfkvl agdknfitae elrrelppdq aeyciarmap yqgpdavpga ldyksfstal
    901 ygesdl
    Activin A receptor type 1, NP_001096.1, NP_001104537.1, NP_001334592.1,
    NP_001334593.1, NP_001334594.1, NP_001334595.1, NP_001334596.1 (SEQ ID NO:
    33)
    1 mvdgvmilpv limialpsps medekpkvnp klymcvcegl scgnedhceg qqcfsslsin
    61 dgfhvyqkgc fqvyeqgkmt cktppspgqa veccqgdwcn rnitaqlptk gksfpgtqnf
    121 hlevgliils vvfavcllac llgvalrkfk rrnqerlnpr dveygtiegl ittnvgdstl
    181 adlldhscts gsgsglpflv qrtvarqitl lecvgkgryg evwrgswqge nvavkifssr
    241 dekswfrete lyntvmlrhe nilgfiasdm tsrhsstqlw lithyhemgs lydylqlttl
    301 dtvsclrivl siasglahlh ieifgtqgkp aiahrdlksk nilvkkngqc ciadlglavm
    361 hsqstnqldv gnnprvgtkr ymapevldet iqvdcfdsyk rvdiwafglv lwevarrmvs
    421 ngivedykpp fydvvpndps fedmrkvvcv dqqrpnipnr wfsdptltsl aklmkecwyq
    481 npsarltalr ikktltkidn sldklktdc
    Alcohol dehydrogenase 1C (class I), gamma polypeptide, NP_000660.1 (SEQ ID
    NO: 34)
    1 mstagkvikc kaavlwelkk pfsieeveva ppkahevrik mvaagicrsd ehvvsgnlvt
    61 plpvilghea agivesvgeg vttvkpgdkv iplftpqcgk cricknpesn yclkndlgnp
    121 rgtlqdgtrr ftcsgkpihh fvgvstfsqy tvvdenavak idaasplekv cligcgfstg
    181 ygsavkvakv tpgstcavfg lggvglsvvm gckaagaari iavdinkdkf akakelgate
    241 cinpqdykkp iqevlkemtd ggvdfsfevi grldtmmasl lccheacgts vivgvppdsq
    301 nlsinpmlll tgrtwkgaif ggfkskesvp klvadfmakk fsldalitni lpfekinegf
    361 dllrsgksir tvltf
    Adenosine A2a receptor, NP_000666.2, NP_001265426.1, NP_001265427.1,
    NP_001265428.1, NP_ 001265429.1 (SEQ ID NO: 35)
    1 mpimgssvyi tvelaiavla ilgnvlvcwa vwlnsnlqnv tnyfvvslaa adiavgvlai
    61 pfaitistgf caachgclfi acfvlvltqs sifsllaiai dryiairipl rynglvtgtr
    121 akgiiaicwv lsfaigltpm lgwnncgqpk egknhsqgcg egqvaclfed vvpmnymvyf
    181 nffacvlvpl llmlgvylri flaarrqlkg mesqplpger arstlqkevh aakslaiivg
    241 lfalcwlplh iincftffcp dcshaplwlm ylaivlshtn svvnpfiyay rirefrqtfr
    301 kiirshvlrq qepfkaagts arvlaahgsd geqvslrlng hppgvwangs aphperrpng
    361 yalglvsggs aqesqgntgl pdvellshel kgvcpeppgl ddplaqdgag vs
    Rho guanine nucleotide exchange factor 16, NP_055263.2 (SEQ ID NO: 36)
    1 magrhsdssl eekllghrfh selrldaggn pasglpmvrg sprvrddaaf qpqvpappqp
    61 rppgheepwp ivlstespaa lklgtqqlip kslavaskak tparhqsfga avlsreaarr
    121 dpkllpapsf slddmdvdkd pggmlrrnlr nqsyraamkg lgkpggqgda iqlspklqal
    181 aeepsqphtr spaknkktlg rkrghkgsfk ddpqlyqeiq erglntsqes dddildesss
    241 pegtqkvdat ivvksyrpaq vtwsqlpevv elgildqlst eerkrqeamf eiltsefsyq
    301 hslsilveef lqskelratv tqmehhhlfs nildvlgasq rffedleqrh kaqvlvedis
    361 dileehaekh fhpyiaycsn evyqqrtlqk lissnaafre alreierrpa cgglpmlsfl
    421 ilpmqrvtrl pllmdtlclk tqghseryka asralkaisk lvrqcnegah rmermeqmyt
    481 lhtqldfskv kslplisasr wllkrgelfl veetglfrki asrptcylfl fndvlvvtkk
    541 kseesymvqd yaqmnhiqve kiepselplp gggnrsssvp hpfqvtllrn segrqeqlll
    601 ssdsasdrar wivalthser qwqglsskgd lpqveitkaf fakqadevtl qqadvvlvlq
    661 qedgwlyger lrdgetgwfp edfarfitsr vavegnvrrm erlrvetdv
    B-cell linker, isoform 1, NP_037446.1 (SEQ ID NO: 37)
    1 mdklnkitvp asqklrqlqk mvhdiknneg gimnkikklk vkappsvprr dyasespade
    61 eeqwsddfds dyenpdehsd semyvmpaee naddsyeppp veqetrpvhp alpfargeyi
    121 dnrssqrhsp pfsktlpskp swpsekarlt stlpaltalq kpqvppkpkg lledeadyvv
    181 pvedndenyi hptesssppp ekapmvnrst kpnsstpasp pgtasgrnsg awetkspppa
    241 apsplpragk kpttplkttp vasqqnassv ceekpipaer hrgsshrqea vqspvfppaq
    301 kqihqkpipl prfteggnpt vdgplpsfss nstiseqeag vlckpwyaga cdrksaeeal
    361 hrsnkdgsfl irkssghdsk qpytlvvffn krvynipvrf ieatkqyalg rkkngeeyfg
    421 svaeiirnhq hsplvlidsq nntkdstrlk yavkvs
    B-cell linker, isoform 2, NP_001107566.1 (SEQ ID NO: 38)
    1 mdklnkitvp asqklrqlqk mvhdiknneg gimnkikklk vkappsvprr dyasespade
    61 eeqwsddfds dyenpdehsd semyvmpaee naddsyeppp veqetrpvhp alpfargeyi
    121 dnrssqrhsp pfsktlpskp swpsekarlt stlpaltalq kpqvppkpkg lledeadyvv
    181 pvedndenyi hptesssppp ekgrnsgawe tkspppaaps plpragkkpt tplkttpvas
    241 qqnassvcee kpipaerhrg sshrqeavqs pvfppaqkqi hqkpiplprf teggnptvdg
    301 plpsfssnst iseqeagvlc kpwyagacdr ksaeealhrs nkdgsflirk ssghdskqpy
    361 tivvffnkrv ynipvrfiea tkqyalgrkk ngeeyfgsva eiirnhqhsp lvlidsqnnt
    421 kdstrlkyav kvs
    B-cell linker, isoform 3, NP_001245369.1 (SEQ ID NO: 39)
    1 mdklnkitvp asqklrqlqk mvhdiknneg gimnkikklk vkappsvprr dyasespade
    61 eeqwsddfds dyenpdehsd semyvmpaee naddsyeppp veqetrpvhp alpfargeyi
    121 dnrssqrhsp pfsktlpskp swpsekarlt stlpaltalq kpqvppkpkg lledeadyvv
    181 pvedndenyi hptesssppp ekapmvnrst kpnsstpasp pgtasgrnsg awetkspppa
    241 apsplpragk kpttplkttp vasqqnassv ceekpipaer hrgsshrqea vqspvfppaq
    301 kqihqkpipl prfteggnpt vdgplpsfss nstiseqeag vlckpwyaga cdrksaeeal
    361 hrsnkyfgsv aeiirnhqhs plvlidsqnn tkdstrlkya vkvs
    B-cell linker, isoform 4, NP_001245370.1 (SEQ ID NO: 40)
    1 mdklnkitvp asqklrqlqk mvhdiknneg gimnkikklk vkappsvprr dyasespade
    61 eeqwsddfds dyenpdehsd semyvmpaee naddsyeppp veqetrpvhp alpfargeyi
    121 dnrssqrhsp pfsktlpskp swpsekarlt stlpaltalq kpqvppkpkg lledeadyvv
    181 pvedndenyi hptesssppp ekgrnsgawe tkspppaaps plpragkkpt tplkttpvas
    241 qqnassvcee kpipaerhrg sshrqeavqs pvfppaqkqi hqkpiplprf teggnptvdg
    301 plpsfssnst iseqeagvlc kpwyagacdr ksaeealhrs nkyfgsvaei irnhqhsplv
    361 lidsqnntkd strlkyavkv s
    B-cell linker, isoform 5, NP_001245371.1 (SEQ ID NO: 41)
    1 mdklnkitvp asqklrqlqk mvhdiknneg gimnkikklk vkappsvprr dyasespade
    61 eeqwsddfds dyenpdehsd semyvmpaee naddsyeppp veqetrpvhp alpfargtas
    121 grnsgawetk spppaapspl pragkkpttp lkttpvasqq nassvceekp ipaerhrgss
    181 hrqeavqspv fppaqkqihq kpiplprfte ggnptvdgpl psfssnstis eqeagvlckp
    241 wyagacdrks aeealhrsnk yfgsvaeiir nhqhsplvli dsqnntkdst rlkyavkvs
    Basonuclin 1, isoform a, NP_001708.3 (SEQ ID NO: 42)
    1 mrrrppsrgg rgaararetr rqprhrsgrr maeaisctln cscqsfkpgk inhrqcdqck
    61 hgwvahalsk lrippmypts qveivqsnvv fdisslmlyg tqaipvrlki lldrlfsvlk
    121 qdevlqilha ldwtlqdyir gyvlqdasgk vldhwsimts eeevatlqqf lrfgetksiv
    181 elmaiqekee qsiiippsta nvdirafies cshrssslpt pvdkgnpssi hpfenlisnm
    241 tfmlpfqffn plppaligsl peqymleqgh dqsqdpkqev hgpfpdssfl tssstpfqve
    301 kdqclncpda itkkedsthl sdsssynivt kfertqlspe akvkpernsl gtkkgrvfct
    361 acektfydkg tlkihynavh lkikhkctie gcnmvfsslr srnrhsanpn prlhmpmnrn
    421 nrdkdlrnsl nlassenykc pgftvtspdc rpppsypgsg edskgqpafp nigqngvlfp
    481 nlktvqpvlp fyrspatpae vantpgilps lpllsssipe qlisnempfd alpkkksrks
    541 smpikiekea veianekrhn lssdedmplq vvsedeqeac spqshrvsee qhvqsgglgk
    601 pfpegerpch resviessga isqtpeqath nsereteqtp alimvpreve dgghehyftp
    661 gmepqvpfsd ymelqqrlla gglfsalsnr gmafpcleds kelehvgqha larqieenrf
    721 qcdickktfk nacsvkihhk nmhvkemhtc tvegcnatfp srrsrdrhss nlnlhqkals
    781 qealessedh fraayllkdv akeayqdvaf tqqasqtsvi fkgtsrmgsl vypitqvhsa
    841 slesynsgpl segtildlst tssmksesss hsswdsdgvs eegtvlmeds dgncegsslv
    901 pgedeypicv lmekadqsla slpsglpitc hlcqktysnk gtfrahyktv hlrqlhkckv
    961 pgcntmfssv rsrnrhsqnp nlhkslassp shlq
    Basonuclin 1, isoform b, NP_001288135.1 (SEQ ID NO: 43)
    1 mrcrnmffsf kaslcgcgaa tapsltaisc tlncscqsfk pgkinhrqcd qckhgwvaha
    61 lsklrippmy ptsqveivqs nvvfdisslm lygtqaipvr lkilldrlfs vlkqdevlqi
    121 lhaldwtlqd yirgyvlqda sgkvldhwsi mtseeevatl qqflrfgetk sivelmaiqe
    181 keeqsiiipp stanvdiraf iescshrsss lptpvdkgnp ssihpfenli snmtfmlpfq
    241 ffnplppali gslpeqymle qghdqsqdpk cqvhgpfpds sfltssstpf qvekdqclnc
    301 pdaitkkeds thlsdsssyn ivtkfertql speakvkper nslgtkkgrv fctacektfy
    361 dkgtlkihyn avhlkikhkc tiegcnmvfs slrsrnrhsa npnprlhmpm nrnnrdkdlr
    421 nslnlassen ykcpgftvts pdcrpppsyp gsgedskgqp afpnigqngv lfpnlktvqp
    481 vlpfyrspat paevantpgi lpslpllsss ipeqlisnem pfdalpkkks rkssmpikie
    541 keaveianek rhnlssdedm plqvvsedeq eacspqshrv seeqhvqsgg lgkpfpeger
    601 pchresvies sgaisqtpeq athnserete qtpalimvpr evedgghehy ftpgmepqvp
    661 fsdymelqqr llagglfsal snrgmafpcl edskelehvg qhalarqiee nrfqcdickk
    721 tfknacsvki hhknmhvkem htctvegcna tfpsrrsrdr hssnlnlhqk alsqealess
    781 edhfraayll kdvakeayqd vaftqqasqt svifkgtsrm gslvypitqv hsaslesyns
    841 gplsegtild lsttssmkse ssshsswdsd gvseegtvlm edsdgncegs slvpgedeyp
    901 icvlmekadq slaslpsglp itchlcqkty snkgtfrahy ktvhlrqlhk ckvpgcntmf
    961 ssvrsrnrhs qnpnlhksla sspshlq
    BPI fold containing family A member 1, precursor, NP_001230122.1,
    NP_057667.1, NP_570913.1 (SEQ ID NO: 44)
    1 mfqtgglivf ygllaqtmaq fgglpvpldq tlplnvnpal plsptglags ltnalsngll
    61 sggllgilen lplldilkpg ggtsggllgg llgkvtsvip glnniidikv tdpqllelgl
    121 vqspdghrly vtiplgiklq vntplvgasl lrlavkldit aeilavrdkq erihlvlgdc
    181 thspgslqis lldglgplpi qglldsltgi lnkvlpelvq gnvcplvnev lrglditivh
    241 divnmlihgl qfvikv
    Calcium voltage-gated channel auxiliary subunit beta 3, isoform 1,
    NP_000716.2 (SEQ ID NO: 45)
    1 myddsyvpgf edseagsads ytsrpsldsd vsleedresa rrevesqaqq qlerakhkpv
    61 afavrtnvsy cgvldeecpv qgsgvnfeak dflhikekys ndwwigrlvk eggdiafips
    121 pqrlesirlk qeqkarrsgn psslsdignr rspppslakq kqkqaehvpp ydvvpsmrpv
    181 vlvgpslkgy evtdmmqkal fdflkhrfdg risitrvtad lslakrsvin npgkrtiier
    241 ssarssiaev qseierifel akslqlvvld adtinhpaql aktslapiiv fvkvsspkvl
    301 qrlirsrgks qmkhltvqmm aydklvqcpp esfdvilden qledacehla eylevywrat
    361 hhpapgpgll gppsaipglq nqqllgerge ehsplerdsl mpsdeasess rqawtgssqr
    421 ssrhleedya dayqdlyqph rqhtsglpsa nghdpqdrll aqdsehnhsd rnwqrnrpwp
    481 kdsy
    Calcium voltage-gated channel auxiliary subunit beta 3, isoform 2,
    NP_001193844.1 (SEQ ID NO: 46)
    1 myddsyvpgf edseagsads ytsrpsldsd vsleedresa rrevesqaqq qlerakkysn
    61 dwwigrlvke ggdiafipsp qrlesirlkq eqkarrsgnp sslsdignrr spppslakqk
    121 qkqaehvppy dvvpsmrpvv lvgpslkgye vtdmmqkalf dflkhrfdgr isitrvtadl
    181 slakrsvlnn pgkrtiiers sarssiaevq seierifela kslqlvvlda dtinhpaqla
    241 ktslapiivf vkvsspkvlq rlirsrgksq mkhltvqmma ydklvqcppe sfdvildenq
    301 ledacehlae ylevywrath hpapgpgllg ppsaipglqn qqllgergee hsplerdslm
    361 psdeasessr qawtgssqrs srhleedyad ayqdlyqphr qhtsglpsan ghdpqdrlla
    421 qdsehnhsdr nwqrnrpwpk dsy
    Calcium voltage-gated channel auxiliary subunit beta 3, isoform 3,
    NP_001193845.1 (SEQ ID NO: 47)
    1 msfsdssatf llnegsadsy tsrpsldsdv sleedresar revesqaqqg lerakhkpva
    61 favrtnvsyc gvldeecpvq gsgvnfeakd flhikekysn dwwigrlvke ggdiafipsp
    121 qrlesirlkq eqkarrsgnp sslsdignrr spppslakqk qkqaehvppy dvvpsmrpvv
    181 lvgpslkgye vtdmmqkalf dflkhrfdgr isitrvtadl slakrsvlnn pgkrtiiers
    241 sarssiaevq seierifela kslqlvvlda dtinhpaqla ktslapiivf vkvsspkvlq
    301 rlirsrgksq mkhltvqmma ydklvqcppe sfdvildenq ledacehlae ylevywrath
    361 hpapgpgllg ppsaipglqn qqllgergee hsplerdslm psdeasessr qawtgssqrs
    421 srhleedyad ayqdlyqphr qhtsglpsan ghdpqdrlla qdsehnhsdr nwqrnrpwpk
    481 dsy
    Calcium voltage-gated channel auxiliary subunit beta 3, isoform 4,
    NP_001193846.1 (SEQ ID NO: 48)
    1 megsadsyts rpsldsdvsl eedresarre vesqaqqqle rakhkpvafa vrtnvsycgv
    61 ldeecpvggs gvnfeakdfl hikekysndw wigrlvkegg diafipspqr lesirlkqeq
    121 karrsgnpss lsdignrrsp ppslakqkqk qaehvppydv vpsmrpvvlv gpslkgyevt
    181 dmmqkalfdf lkhrfdgris itrvtadlsl akrsvlnnpg krtiierssa rssiaevqse
    241 ierifelaks lqlvvldadt inhpaglakt slapiivfvk vsspkvlqrl irsrgksqmk
    301 hltvqmmayd klvqcppesf dvildenqle dacehlaeyl evywrathhp apgpgllgpp
    361 saipglqnqq llgergeehs plerdslmps deasessrqa wtgssqrssr hleedyaday
    421 qdlyqphrqh tsglpsangh dpqdrllaqd sehnhsdrnw qrnrpwpkds y
    Caspase 3, preproprotein, NP_ 001341706.1, NP_001341707.1, NP_004346.3,
    NP_116786.1 (SEQ ID NO: 49)
    1 mentensvds ksiknlepki ihgsesmdsg isldnsykmd ypemglciii nnknfhkstg
    61 mtsrsgtdvd aanlretfrn lkyevrnknd ltreeivelm rdvskedhsk rssfvcvlls
    121 hgeegiifgt ngpvdlkkit nffrgdrcrs ltgkpklfii qacrgteldc gietdsgvdd
    181 dmachkipve adflyaysta pgyyswrnsk dgswfiqslc amlkqyadkl efmhiltrvn
    241 rkvatefesf sfdatfhakk qipcivsmlt kelyfyh
    Caspase 3, isoform b, NP_001341708.1, NP_001341709.1 (SEQ ID NO: 50)
    1 mdsgisldns ykmdypemgl ciiinnknfh kstgmtsrsg tdvdaanlre tfrnlkyevr
    61 nkndltreei velmrdvske dhskrssfvc vllshgeegi ifgtngpvdl kkitnffrgd
    121 rcrsltgkpk lfiiqacrgt eldcgietds gvdddmachk ipveadflya ystapgyysw
    181 rnskdgswfi qslcamlkqy adklefmhil trvnrkvate fesfsfdatf hakkgipciv
    241 smltkelyfy h
    Caspase 3, isoform c, NP_001341710.1, NP_001341711.1 (SEQ ID NO: 51)
    1 mentensvds ksiknlepki ihgsesmdsg isldnsykmd ypemglciii nnknfhkstg
    61 mtsrsgtdvd aanlretfrn lkyevrnknd ltreeivelm rdvskedhsk rssfvcvlls
    121 hgeegiifgt ngpvdlkkit nffrgdrcrs ltgkpklfii qviilgeiqr mapgsssrfv
    181 pc
    Caspase 3, isoform d, NP_001341712.1 (SEQ ID NO: 52)
    1 msdalikvsm entensvdsk siknlepkii hgsesmdsgi sldnsykmdy pemglciiin
    61 nknfhkstgm tsrsgtdvda anlretfrnl kyevrnkndl treeivelmr dvskedhskr
    121 ssfvcvllsh geegiifgtn gpvdlkkitn ffrgdrcrsl tgkpklfiiq viilgeiqrm
    181 apgsssrfvp c
    Caspase 3, isoform e, NP_001341713.1 (SEQ ID NO: 53)
    1 mdsgisldns ykmdypemgl ciiinnknfh kstgmtsrsg tdvdaanlre tfrnlkyevr
    61 nkndltreei velmrdvske dhskrssfvc vllshgeegi ifgtngpvdl kkitnffrgd
    121 rcrsltgkpk lfiiqviilg eiqrmapgss srfvpc
    Caveolin 1, isoform alpha, NP_001744.2 (SEQ ID NO: 54)
    1 msggkyvdse ghlytvpire qgniykpnnk amadelsekq vydahtkeid lvnrdpkhln
    61 ddvvkidfed viaepegths fdgiwkasft tftvtkywfy rllsalfgip maliwgiyfa
    121 ilsflhiwav vpciksflie iqcisrvysi yvhtvcdplf eavgkifsnv rinlqkei
    Caveolin 1, isoform beta, NP_001166366.1, NP_001166367.1, NP_001166368.1 (SEQ
    ID NO: 55)
    1 madelsekqv ydahtkeidl vnrdpkhlnd dvvkidfedv iaepegthsf dgiwkasftt
    61 ftvtkywfyr llsalfgipm aliwgiyfai lsflhiwavv pciksfliei qcisrvysiy
    121 vhtvcdplfe avgkifsnvr inlqkei
    Cadherin 1, isoform 1 preproprotein, NP_004351.1 (SEQ ID NO: 56)
    1 mgpwsrslsa lllllqvssw logepepchp gfdaesytft vprrhlergr vlgrvnfedc
    61 tgrqrtayfs ldtrfkvgtd gvitvkrplr fhnpqihflv yawdstyrkf stkvtlntvg
    121 hhhrppphqa sysgiqaell tfpnsspglr rqkrdwvipp iscpenekgp fpknlvqiks
    181 nkdkegkvfy sitgqgadtp pvgvfiiere tgwlkvtepl dreriatytl fshayssngn
    241 avedpmeili tvtdqndnkp eftqevfkgs vmegalpgts vmevtatdad ddvntynaai
    301 aytilsqdpe lpdknmftin rntgvisvvt tgldresfpt ytlvvqaadl qgeglsttat
    361 avitvtdtnd nppifnptty kgqvpenean vvittlkvtd adapntpawe avytilnddg
    421 gqfvvttnpv nndgilktak gldfeakqqy ilhvavtnvv pfevslttst atvtvdvldv
    481 neapifvppe krvevsedfg vggeitsyta qepdtfmeqk ityriwrdta nwleinpdtg
    541 aistraeldr edfehvknst ytaliiatdn gspvatgtgt lllilsdvnd napipeprti
    601 ffcernpkpq viniidadlp pntspftael thgasanwti qyndptqesi ilkpkmalev
    661 gdykinlklm dnqnkdqvtt levsvcdceg aagvcrkaqp veaglqipai lgilggilal
    721 lililllllf lrrravvkep llppeddtrd nvyyydeegg geedqdfdls qlhrgldarp
    781 evtrndvapt lmsvprylpr panpdeignf idenlkaadt dptappydsl lvfdyegsgs
    841 eaaslsslns sesdkdqdyd ylnewgnrfk kladmyggge dd
    Cadherin 1, isoform 2 precursor, NP_001304113.1 (SEQ ID NO: 57)
    1 mgpwsrslsa lllllqvssw lcqepepchp gfdaesytft vprrhlergr vlgrvnfedc
    61 tgrqrtayfs ldtrfkvgtd gvitvkrplr fhnpqihflv yawdstyrkf stkvtlntvg
    121 hhhrppphqa sysgiqaell tfpnsspglr rqkrdwvipp iscpenekgp fpknlvqiks
    181 nkdkegkvfy sitgqgadtp pvgvfiiere tgwlkvtepl dreriatytl fshayssngn
    241 avedpmeili tvtdqndnkp eftqevfkgs vmegalpgts vmevtatdad ddvntynaai
    301 aytilsqdpe lpdknmftin rntgvisvvt tgldresfpt ytlvvqaadl qgeglsttat
    361 avitvtdtnd nppifnpttg ldfeakqqyi lhvavtnvvp fevslttsta tvtvdvldvn
    421 eapifvppek rvevsedfgv gqeitsytaq epdtfmeqki tyriwrdtan wleinpdtga
    481 istraeldre dfehvknsty taliiatdng spvatgtgtl llilsdvndn apipeprtif
    541 fcernpkpqv iniidadlpp ntspftaelt hgasanwtiq yndptqesii lkpkmalevg
    601 dykinlklmd nqnkdqvttl evsvcdcega agvcrkaqpv eaglqipail gilggilall
    661 ililllllfl rrravvkepl lppeddtrdn vyyydeeggg eedqdfdlsq lhrgldarpe
    721 vtrndvaptl msvprylprp anpdeignfi denlkaadtd ptappydsll vfdyegsgse
    781 aaslsslnss esdkdqdydy lnewgnrfkk ladmyggged d
    Cadherin 1, isoform 3, NP_001304114.1 (SEQ ID NO: 58)
    1 meqkityriw rdtanwlein pdtgaistra eldredfehv knstytalii atdngspvat
    61 gtgtlllils dvndnapipe prtiffcern pkpqviniid adlppntspf taelthgasa
    121 nwtiqyndpt qesiilkpkm alevgdykin lklmdnqnkd qvttlevsvc dcegaagvcr
    181 kaqpveaglq ipailgilgg ilallilill lllflrrrav vkepllpped dtrdnvyyyd
    241 eegggeedqd fdlsqlhrgl darpevtrnd vaptlmsvpr ylprpanpde ignfidenlk
    301 aadtdptapp ydsllvfdye gsgseaasls slnssesdkd qdydylnewg nrfkkladmy
    361 gggedd
    Cadherin 1, isoform 4, NP_001304115.1 (SEQ ID NO: 59)
    1 malevgdyki nlklmdnqnk dqvttlevsv cdcegaagvc rkaqpveagl qipailgilg
    61 gilallilil llllflrrra vvkepllppe ddtrdnvyyy deegggeedq dfdlsqlhrg
    121 ldarpevtrn dvaptlmsvp rylprpanpd eignfidenl kaadtdptap pydsllvfdy
    181 egsgseaasl sslnssesdk dqdydylnew gnrfkkladm ygggedd
    Cytochrome c oxidase subunit 8C, NP_892016.1 (SEQ ID NO: 60)
    1 mpllrgrcpa rrhyrrlall glqpaprfah sgpprqrpls aaemavglvv ffttfltpaa
    61 yvlgnlkqfr rn
    Carnitine palmitoyltransferase 1A, isoform 1, NP_001867.2 (SEQ ID NO: 61)
    1 maeahqavaf qftvtpdgid lrlshealrq iylsglhswk kkfirfkngi itgvypasps
    61 swlivvvgvm ttmyakidps lgiiakinrt letancmssq tknvvsgvlf gtglwvaliv
    121 tmryslkvll syhgwmfteh gkmsratkiw mgmvkifsgr kpmlysfqts lprlpvpavk
    181 dtvnrylqsv rplmkeedfk rmtalaqdfa vglgprlqwy lklkswwatn yvsdwweeyi
    241 ylrgrgplmv nsnyyamdll yilpthiqaa ragnaihail lyrrkldree ikpirllgst
    301 iplcsaqwer mfntsripge etdtiqhmrd skhivvyhrg ryfkvwlyhd grllkpreme
    361 qqmqrildnt sepqpgearl aaltagdrvp warcrqayfg rgknkqslda vekaaffvtl
    421 deteegyrse dpdtsmdsya ksllhgrcyd rwfdksftfv vfkngkmgln aehswadapi
    481 vahlweyvms idslqlgyae dghckgdinp nipyptrlqw dipgecqevi etslntanll
    541 andvdfhsfp fvafgkgiik kcrtspdafv qlalqlahyk dmgkfcltye asmtrlfreg
    601 rtetvrsctt escdfvramv dpaqtveqrl klfklasekh qhmyrlamtg sgidrhlfcl
    661 yvvskylave spflkevlse pwrlstsqtp qqqvelfdle nnpeyvssgg gfgpvaddgy
    721 gvsyilvgen linfhisskf scpetdshrf grhlkeamtd iitlfglssn skk
    Carnitine palmitoyltransferase 1A, isoform 2, NP_001027017.1 (SEQ ID NO: 62)
    1 maeahqavaf qftvtpdgid lrlshealrq iylsglhswk kkfirfkngi itgvypasps
    61 swlivvvgvm ttmyakidps lgiiakinrt letancmssq tknvvsgvlf gtglwvaliv
    121 tmryslkvll syhgwmfteh gkmsratkiw mgmvkifsgr kpmlysfqts lprlpvpavk
    181 dtvnrylqsv rplmkeedfk rmtalaqdfa vglgprlqwy lklkswwatn yvsdwweeyi
    241 ylrgrgplmv nsnyyamdll yilpthiqaa ragnaihail lyrrkldree ikpirllgst
    301 iplcsaqwer mfntsripge etdtiqhmrd skhivvyhrg ryfkvwlyhd grllkpreme
    361 qqmqrildnt sepqpgearl aaltagdrvp warcrqayfg rgknkqslda vekaaffvtl
    421 deteegyrse dpdtsmdsya ksllhgrcyd rwfdksftfv vfkngkmgln aehswadapi
    481 vahlweyvms idslqlgyae dghckgdinp nipyptrlqw dipgecqevi etslntanll
    541 andvdfhsfp fvafgkgiik kcrtspdafv qlalqlahyk dmgkfcltye asmtrlfreg
    601 rtetvrsctt escdfvramv dpaqtveqrl klfklasekh qhmyrlamtg sgidrhlfcl
    661 yvvskylave spflkevlse pwrlstsqtp qqqvelfdle nnpeyvssgg gfgpvaddgy
    721 gvsyilvgen linfhisskf scpetgiisq gpssdt
    Cancer/testis antigen 1A, NP_640343.1 (SEQ ID NO: 63)
    1 mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgaga arasgpggga
    61 prgphggaas glngccrcga rgpesrllef ylampfatpm eaelarrsla qdapplpvpg
    121 vllkeftvsg niltirltaa dhrqlqlsis sclqqlsllm witqcflpvf laqppsgqrr
    C-X-C motif chemokine ligand 13, NP_006410.1 (SEQ ID NO: 64)
    1 mkfistslll mllvsslspv qgvlevyyts lrcrcvqess vfiprrfidr iqilprgngc
    61 prkeiivwkk nksivcvdpq aewiqrmmev lrkrssstlp vpvfkrkip
    Diacylglycerol kinase eta, isoform 1, NP_001191433.1, NP_690874.2 (SEQ ID NO:
    65)
    1 magaggqhhp pgaaggaaag agaavtsaaa sagpgedssd seaeqegpqk lirkvstsgq
    61 irtktsikeg qllkqtssfq rwkkryfklr grtlyyakds kslifdevdl sdasvaeast
    121 knannsftii tpfrrlmlca enrkemedwi sslksvqtre pyevaqfnve hfsgmhnwya
    181 csharptfcn vcreslsgvt shglscevck fkahkrcavr atnnckwttl asigkdiied
    241 edgvamphqw legnlpvsak cavcdktcgs vlrlqdwkcl wcktmvhtac kdlyhpicpl
    301 gqckvsiipp ialnstdsdg fcratfsfcv spllvfvnsk sgdnqgvkfl rrfkqllnpa
    361 qvfdlmnggp hlglrlfqkf dnfrilvcgg dgsvgwvlse idklnlnkqc qlgvlplgtg
    421 ndlarvlgwg gsydddtqlp qilekleras tkmldrwsim tyelklppka sllpgppeas
    481 eefymtiyed svathltkil nsdehavvis saktlcetvk dfvakvekty dktlenavva
    541 davaskcsvl nekleqllqa lhtdsqaapv lpglsplive edavesssee slgeskeqlg
    601 ddvtkpssqk avkpreimlr anslkkavrq vieeagkvmd dptvhpcepa nqssdydste
    661 tdeskeeakd dgakesitvk taprspdara syghsqtdsv pgpavaaske nlpvintrii
    721 cpglraglaa siagssiink mllanidpfg atpfidpdld svdgysekcv mnnyfgigld
    781 akislefnnk reehpekcrs rtknlmwygv lgtrellqrs yknleqrvql ecdgqyiplp
    841 slqgiavini psyaggtnfw ggtkeddifa apsfddkile vvaifdsmqm aysrviklqh
    901 hriaqcrtvk itifgdegvp vqvdgeawvq ppgiikivhk nraqmltrdr afestlkswe
    961 dkqkcdsgkp vlrthlyihh aidlateevs qmqlcsqaae elitricdaa tihclleqel
    1021 ahavnacsha lnkanprcpe sltrdtatei ainvkalyne tesllvgrvp lqlespheer
    1081 vsnalhsvev elqklteipw lyyilhpned eeppmdctkr nnrstvfriv pkfkkekvqk
    1141 qktssqpgsg dtesgscean spgn
    Diacylglycerol kinase eta, isoform 2, NP_821077.1 (SEQ ID NO: 66)
    1 magaggqhhp pgaaggaaag agaavtsaaa sagpgedssd seaeqegpqk lirkvstsgq
    61 irtktsikeg qllkqtssfq rwkkryfklr grtlyyakds kslifdevdl sdasvaeast
    121 knannsftii tpfrrlmlca enrkemedwi sslksvqtre pyevaqfnve hfsgmhnwya
    181 csharptfcn vcreslsgvt shglscevck fkahkrcavr atnnckwttl asigkdiied
    241 edgvamphqw legnlpvsak cavcdktcgs vlrlqdwkcl wcktmvhtac kdlyhpicpl
    301 gqckvsiipp ialnstdsdg fcratfsfcv spllvfvnsk sgdnqgvkfl rrfkqllnpa
    361 qvfdlmnggp hlglrlfqkf dnfrilvcgg dgsvgwvlse idklnlnkqc qlgvlplgtg
    421 ndlarvlgwg gsydddtqlp qilekleras tkmldrwsim tyelklppka sllpgppeas
    481 eefymtiyed svathltkil nsdehavvis saktlcetvk dfvakvekty dktlenavva
    541 davaskcsvl nekleqllqa lhtdsqaapv lpglsplive edavesssee slgeskeqlg
    601 ddvtkpssqk avkpreimlr anslkkavrq vieeagkvmd dptvhpcepa nqssdydste
    661 tdeskeeakd dgakesitvk taprspdara syghsqtdsv pgpavaaske nlpvlntrii
    721 cpglraglaa siagssiink mllanidpfg atpfidpdld svdgysekcv mnnyfgigld
    781 akislefnnk reehpekcrs rtknlmwygv lgtrellqrs yknleqrvql ecdgqyiplp
    841 slqgiavini psyaggtnfw ggtkeddifa apsfddkile vvaifdsmqm aysrviklqh
    901 hriaqcrtvk itifgdegvp vqvdgeawvq ppgiikivhk nraqmltrdr afestlkswe
    961 dkqkcdsgkp vlrthlyihh aidlateevs qmqlcsqaae elitricdaa tihclleqel
    1021 ahavnacsha lnkanprcpe sltrdtatei ainvkalyne tesllvgrvp lqlespheer
    1081 vsnalhsvev elqklteipw lyyilhpned eeppmdctkr nnrstvfriv pkfkkekvqk
    1141 qktssqpvqk wgteevaawl dllnlgeykd ifirhdirga ellhlerrdl kdlgipkvgh
    1201 vkrilqgike lgrstpqsev
    Diacylglycerol kinase eta, isoform 3, NP_001191434.1 (SEQ ID NO: 67)
    1 mlcaenrkem edwisslksv qtrepyevaq fnvehfsgmh nwyacsharp tfcnvcresl
    61 sgvtshglsc evckfkahkr cavratnnck wttlasigkd iiededgvam phqwlegnlp
    121 vsakcavcdk tcgsvlrlqd wkclwcktmv htackdlyhp icplgqckvs iippialnst
    181 dsdgfcratf sfcvspllvf vnsksgdnqg vkflrrfkql lnpaqvfdlm nggphlglrl
    241 fqkfdnfril vcggdgsvgw vlseidklnl nkqcqlgvlp lgtgndlarv lgwggsyddd
    301 tqlpqilekl erastkmldr wsimtyelkl ppkasllpgp peaseefymt iyedsvathl
    361 tkilnsdeha vvissaktic etvkdfvakv ektydktlen avvadavask csvlnekleq
    421 llqalhtdsq aapvlpglsp liveedaves sseeslgesk eqlgddvtkp ssqkavkpre
    481 imlranslkk avrqvieeag kvmddptvhp cepanqssdy dstetdeske eakddgakes
    541 itvktaprsp darasyghsq tdsvpgpava askenlpvin triicpglra glaasiagss
    601 iinkmllani dpfgatpfid pdldsvdgys ekcvmnnyfg igldakisle fnnkreehpe
    661 kcrsrtknlm wygvlgtrel lqrsyknleq rvqlecdgqy iplpslqgia vlnipsyagg
    721 tnfwggtked difaapsfdd kilevvaifd smqmavsrvi klqhhriaqc rtvkitifgd
    781 egvpvqvdge awvqppgiik ivhknraqml trdrafestl kswedkqkcd sgkpvlrthl
    841 yihhaidlat eevsqmqlcs qaaeelitri cdaatihcll eqelahavna cshalnkanp
    901 rcpesltrdt ateiainvka lynetesllv grvplqlesp heervsnalh svevelqklt
    961 eipwlyyilh pnedeeppmd ctkrnnrstv frivpkfkke kvqkqktssq pvqkwgteev
    1021 aawldllnlg eykdifirhd irgaellhle rrdlkntvge krdtkengkh mdlgipkvgh
    1081 vkrilqgike lgrstpqsev
    Diacylglycerol kinase eta, isoform 4, NP_001191435.1 (SEQ ID NO: 68)
    1 mlcaenrkem edwisslksv qtrepyevaq fnvehfsgmh nwyacsharp tfcnvcresl
    61 sgvtshglsc evckfkahkr cavratnnck wttlasigkd iiededgvam phqwlegnlp
    121 vsakcavcdk tcgsvlrlqd wkclwcktmv htackdlyhp icplgqckvs iippialnst
    181 dsdgfcratf sfcvspllvf vnsksgdnqg vkflrrfkql lnpaqvfdlm nggphlglrl
    241 fqkfdnfril vcggdgsvgw vlseidklnl nkqcqlgvlp lgtgndlarv lgwggsyddd
    301 tqlpqilekl erastkmldr wsimtyelkl ppkasllpgp peaseefymt iyedsvathl
    361 tkilnsdeha vvissaktlc etvkdfvakv ektydktlen avvadavask csvlnekleq
    421 llqalhtdsq aapvlpglsp liveedaves sseeslgesk eqlgddvtkp ssqkavkpre
    481 imlranslkk avrqvieeag kvmddptvhp cepanqssdy dstetdeske eakddgakes
    541 itvktaprsp darasyghsq tdsvpgpava askenlpvln triicpglra glaasiagss
    601 iinkmllani dpfgatpfid pdldsvdgys ekcvmnnyfg igldakisle fnnkreehpe
    661 kcrsrtknlm wygvlgtrel lqrsyknleq rvqlecdgqy iplpslqgia vlnipsyagg
    721 tnfwggtked difaapsfdd kilevvaifd smqmavsrvi klqhhriaqc rtvkitifgd
    781 egvpvqvdge awvqppgiik ivhknraqml trdrafestl kswedkqkcd sgkpvlrthl
    841 yihhaidlat eevsgmqlcs qaaeelitri cdaatihcll eqelahavna cshalnkanp
    901 rcpesltrdt ateiainvka lynetesllv grvplqlesp heervsnalh svevelqklt
    961 eipwlyyilh pnedeeppmd ctkrnnrstv frivpkfkke kvqkqktssq pvqkwgteev
    1021 aawldllnlg eykdifirhd irgaellhle rrdlkdlgip kvghvkrilq gikelgrstp
    1081 qsev
    Diacylglycerol kinase eta, isoform 5, NP_001284358.1 (SEQ ID NO: 69)
    1 mwnisqgctt gtpaptpdpp svtcaervfl esppmacpak vhtackdlyh picplgqckv
    61 siippialns tdsdgfcrat fsfcvspllv fvnsksgdnq gvkflrrfkq llnpaqvfdl
    121 mnggphlglr lfqkfdnfri lvcggdgsvg wvlseidkln lnkqcqlgvl plgtgndlar
    181 vlgwggsydd dtqlpqilek lerastkmld rwsimtyelk lppkasllpg ppeaseefym
    241 tiyedsvath ltkilnsdeh avvissaktl cetvkdfvak vektydktle navvadavas
    301 kcsvlnekle qllqalhtds qaapvlpgls pliveedave ssseeslges keqlgddvtk
    361 pssqkavkpr eimlranslk kavrqvieea gkvmddptvh pcepanqssd ydstetdesk
    421 eeakddgake sitvktaprs pdarasyghs qtdsvpgpav aaskenlpvl ntriicpglr
    481 aglaasiags siinkmllan idpfgatpfi dpdldsvdgy sekcvmnnyf gigldakisl
    541 efnnkreehp ekcrsrtknl mwygvlgtre llqrsyknle qrvqlecdgq yiplpslqgi
    601 avinipsyag gtnfwggtke ddifaapsfd dkilevvaif dsmqmavsrv iklqhhriaq
    661 crtvkitifg degvpvqvdg eawvqppgii kivhknraqm ltrdrafest lkswedkqkc
    721 dsgkpvlrth lyihhaidla teevsqmqlc sqaaeelitr icdaatihcl leqelahavn
    781 acshalnkan prcpesltrd tateiainvk alynetesll vgrvplqles pheervsnal
    841 hsvevelqkl teipwlyyil hpnedeeppm dctkrnnrst vfrivpkfkk ekvqkqktss
    901 qpgsgdtesg sceanspgn
    Eukaryotic translation elongation factor 2, NP_001952.1 (SEQ ID NO: 70)
    1 mvnftvdqir aimdkkanir nmsviahvdh gkstltdslv ckagiiasar agetrftdtr
    61 kdeqerciti kstaislfye lsendlnfik qskdgagfli nlidspghvd fssevtaalr
    121 vtdgalvvvd cvsgvcvqte tvlrqaiaer ikpvlmmnkm drallelqle peelyqtfqr
    181 ivenvnviis tygegesgpm gnimidpvlg tvgfgsglhg waftlkqfae myvakfaakg
    241 egqlgpaera kkvedmmkkl wgdryfdpan gkfsksatsp egkklprtfc qlildpifkv
    301 fdaimnfkke etakliekld ikldsedkdk egkpllkavm rrwlpagdal lqmitihlps
    361 pvtaqkyrce llyegppdde aamgikscdp kgplmmyisk mvptsdkgrf yafgrvfsgl
    421 vstglkvrim gpnytpgkke dlylkpiqrt ilmmgryvep iedvpcgniv glvgvdqflv
    481 ktgtittfeh ahnmrvmkfs vspvvrvave aknpadlpkl veglkrlaks dpmvqciiee
    541 sgehiiagag elhleiclkd leedhacipi kksdpvvsyr etvseesnvl clskspnkhn
    601 rlymkarpfp dglaedidkg evsarqelkq rarylaekye wdvaearkiw cfgpdgtgpn
    661 iltditkgvq ylneikdsvv agfqwatkeg alceenmrgv rfdvhdvtlh adaihrgggq
    721 iiptarrcly asvltaqprl mepiylveiq cpeqvvggiy gvlnrkrghv feesqvagtp
    781 mfvvkaylpv nesfgftadl rsntggqafp qcvfdhwqil pgdpfdnssr psqvvaetrk
    841 rkglkegipa ldnfldkl
    Eukaryotic translation initiation factor 5A, isoform A, NP_001137232.1 (SEQ
    ID NO: 71)
    1 mcgtggtdsk trrpphrasf lkrleskplk maddldfetg dagasatfpm qcsalrkngf
    61 vvlkgrpcki vemstsktgk hghakvhlvg idiftgkkye dicpsthnmd vpnikrndfq
    121 ligiqdgyls llqdsgevre dlrlpegdlg keieqkydcg eeilitvlsa mteeaavaik
    181 amak
    Eukaryotic translation initiation factor 5A, isoform B, NP_001137233.1,
    NP_001137234.1, NP_001961.1 (SEQ ID NO: 72)
    1 maddldfetg dagasatfpm qcsalrkngf vvlkgrpcki vemstsktgk hghakvhlvg
    61 idiftgkkye dicpsthnmd vpnikrndfq ligiqdgyls llqdsgevre dlrlpegdlg
    121 keieqkydcg eeilitvlsa mteeaavaik amak
    Fibronectin 1, isoform 1 precursor, NP_997647.1 (SEQ ID NO: 73)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveengest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 ragitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipevpql tdlsfvditd ssiglrwtpl nsstiigyri tvvaagegip ifedfvdssv
    1321 gyytvtglep gidydisvit linggesapt tltqqtavpp ptdlrftnig pdtmrvtwap
    1381 ppsidltnfl vryspvknee dvaelsisps dnavvltnll pgteyvvsys svyeqhestp
    1441 lrgrqktgld sptgidfsdi tansftvhwi apratitgyr irhhpehfsg rpredrvphs
    1501 rnsitltnlt pgteyvvsiv alngreespl ligqqstvsd vprdlevvaa tptslliswd
    1561 apavtvryyr itygetggns pvqeftvpgs kstatisglk pgvdytitvy avtgrgdspa
    1621 sskpisinyr teidkpsqmq vtdvqdnsis vkwlpssspv tgyrvtttpk ngpgptktkt
    1681 agpdqtemti eglqptveyv vsvyaqnpsg esqplvqtav tnidrpkgla ftdvdvdsik
    1741 iawespqgqv sryrvtyssp edgihelfpa pdgeedtael qglrpgseyt vsvvalhddm
    1801 esqpligtqs taipaptdlk ftqvtptsls aqwtppnvql tgyrvrvtpk ektgpmkein
    1861 lapdsssvvv sglmvatkye vsvyalkdtl tsrpaqgvvt tlenvspprr arvtdatett
    1921 itiswrtkte titgfqvdav pangqtpiqr tikpdvrsyt itglqpgtdy kiylytlndn
    1981 arsspvvida staidapsnl rflattpnsl lvswqpprar itgyiikyek pgspprevvp
    2041 rprpgvteat itglepgtey tiyvialknn qksepligrk ktdelpqlvt lphpnlhgpe
    2101 ildvpstvqk tpfvthpgyd tgngiqlpgt sgqgpsvgqq mifeehgfrr ttppttatpi
    2161 rhrprpyppn vgeeiqighi predvdyhly phgpglnpna stgqealsqt tiswapfqdt
    2221 seyiischpv gtdeeplqfr vpgtstsatl tgltrgatyn iivealkdqq rhkvreevvt
    2281 vgnsvnegln qptddscfdp ytvshyavgd ewermsesgf kllcqclgfg sghfrcdssr
    2341 wchdngvnyk igekwdrqge ngqmmsctcl gngkgefkcd pheatcyddg ktyhvgeqwq
    2401 keylgaicsc tcfggqrgwr cdncrrpgge pspegttgqs ynqysqryhq rtntnvncpi
    2461 ecfmpldvqa dredsre
    Fibronectin 1, isoform 3 precursor, NP_002017.1 (SEQ ID NO: 74)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsysk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd
    1321 navvltnllp gteyvvsyss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia
    1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll
    1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk
    1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvgdnsisv
    1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge
    1621 sqplvqtavt nidrpkglaf tdvdvdsiki awespqgqvs ryrvtysspe dgihelfpap
    1681 dgeedtaelq glrpgseytv svvalhddme sqpligtqst aipaptdlkf tqvtptslsa
    1741 qwtppnvqlt gyrvrvtpke ktgpmkeinl apdsssvvvs glmvatkyev svyalkdtlt
    1801 srpaqgvvtt lenvspprra rvtdatetti tiswrtktet itgfqvdavp angqtpiqrt
    1861 ikpdvrsyti tglqpgtdyk iylytlndna rsspvvidas taidapsnlr flattpnsll
    1921 vswqpprari tgyiikyekp gspprevvpr prpgvteati tglepgteyt iyvialknnq
    1981 ksepligrkk tdelpqlvtl phpnlhgpei ldvpstvqkt pfvthpgydt gngiqlpgts
    2041 gqqpsvgqqm ifeehgfrrt tppttatpir hrprpyppnv gqealsqtti swapfqdtse
    2101 yiischpvgt deeplqfrvp gtstsatltg ltrgatynii vealkdqqrh kvreevvtvg
    2161 nsvneglnqp tddscfdpyt vshyavgdew ermsesgfkl lcqclgfgsg hfrcdssrwc
    2221 hdngvnykig ekwdrqgeng qmmsctclgn gkgefkcdph eatcyddgkt yhvgeqwqke
    2281 ylgaicsctc fggqrgwrcd ncrrpggeps pegttgqsyn qysqryhqrt ntnvncpiec
    2341 fmpldvqadr edsre
    Fibronectin 1, isoform 4 precursor, NP_997643.1 (SEQ ID NO: 75)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvavsqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd
    1321 navvltnllp gteyvvsyss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia
    1381 pratitgyri rhhpehfsgr predrvphsr nsitltnitp gteyvvsiva lngreespll
    1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk
    1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv
    1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge
    1621 sqplvqtavt nidrpkglaf tdvdvdsiki awespqgqvs ryrvtysspe dgihelfpap
    1681 dgeedtaelq glrpgseytv svvalhddme sqpligtqst aipaptdlkf tqvtptslsa
    1741 qwtppnvqlt gyrvrvtpke ktgpmkeinl apdsssvvvs glmvatkyev svyalkdtlt
    1801 srpaqgvvtt lenvspprra rvtdatetti tiswrtktet itgfqvdavp angqtpiqrt
    1861 ikpdvrsyti tglqpgtdyk iylytlndna rsspvvidas taidapsnlr flattpnsll
    1921 vswqpprari tgyiikyekp gspprevvpr prpgvteati tglepgteyt iyvialknnq
    1981 ksepligrkk tvqktpfvth pgydtgngiq lpgtsgqqps vgqqmifeeh gfrrttpptt
    2041 atpirhrprp yppnvgqeal sqttiswapf qdtseyiisc hpvgtdeepl qfrvpgtsts
    2101 atltgltrga tyniivealk dqqrhkvree vvtvgnsvne glnqptddsc fdpytvshya
    2161 vgdewermse sgfkllcgcl gfgsghfrcd ssrwchdngv nykigekwdr qgengqmmsc
    2221 tclgngkgef kcdpheatcy ddgktyhvge qwgkeylgai csctcfggqr gwrcdncrrp
    2281 ggepspegtt gqsynqysqr yhqrtntnvn cpiecfmpld vqadredsre
    Fibronectin 1, isoform 5 precursor, NP_997641.1 (SEQ ID NO: 76)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsysk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd
    1321 navvltnllp gteyvvsvss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia
    1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll
    1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk
    1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvgdnsisv
    1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge
    1621 sqplvqtavt tipaptdlkf tqvtptslsa qwtppnvqlt gyrvrvtpke ktgpmkeinl
    1681 apdsssvvvs glmvatkyev svyalkdtlt srpaqgvvtt lenvspprra rvtdatetti
    1741 tiswrtktet itgfqvdavp angqtpiqrt ikpdvrsyti tglqpgtdyk iylytlndna
    1801 rsspvvidas taidapsnlr flattpnsll vswqpprari tgyiikyekp gspprevvpr
    1861 prpgvteati tglepgteyt iyvialknnq ksepligrkk tdelpqlvtl phpnlhgpei
    1921 ldvpstvqkt pfvthpgydt gngiqlpgts gqqpsvgqqm ifeehgfrrt tppttatpir
    1981 hrprpyppnv geeiqighip redvdyhlyp hgpglnpnas tgqealsqtt iswapfqdts
    2041 eyiischpvg tdeeplqfrv pgtstsatlt gltrgatyni ivealkdqqr hkvreevvtv
    2101 gnsvneglnq ptddscfdpy tvshyavgde wermsesgfk llcqclgfgs ghfrcdssrw
    2161 chdngvnyki gekwdrqgen gqmmsctclg ngkgefkcdp heatcyddgk tyhvgeqwqk
    2221 eylgaicsct cfggqrgwrc dncrrpggep spegttgqsy nqysqryhqr tntnvncpie
    2281 cfmpldvqad redsre
    Fibronectin 1, isoform 6 precursor, NP_997639.1 (SEQ ID NO: 77)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsvsk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd
    1321 navvltnllp gteyvvsyss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia
    1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll
    1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk
    1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv
    1561 kwlpssspvt gvrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge
    1621 sqplvqtavt tipaptdlkf tqvtptslsa qwtppnvqlt gyrvrvtpke ktgpmkeinl
    1681 apdsssvvvs glmvatkyev svyalkdtlt srpaqgvvtt lenvspprra rvtdatetti
    1741 tiswrtktet itgfqvdavp angqtpiqrt ikpdvrsyti tglqpgtdyk iylytlndna
    1801 rsspvvidas taidapsnlr flattpnsll vswqpprari tgyiikyekp gspprevvpr
    1861 prpgvteati tglepgteyt iyvialknnq ksepligrkk tgqealsqtt iswapfqdts
    1921 eyiischpvg tdeeplqfrv pgtstsatlt gltrgatyni ivealkdqqr hkvreevvtv
    1981 gnsvneglnq ptddscfdpy tvshyavgde wermsesgfk llcqclgfgs ghfrcdssrw
    2041 chdngvnyki gekwdrqgen gqmmsctclg ngkgefkcdp heatcyddgk tyhvgeqwqk
    2101 eylgaicsct cfggqrgwrc dncrrpggep spegttgqsy nqysqryhqr tntnvncpie
    2161 cfmpldvqad redsre
    Fibronectin 1, isoform 7 precursor, NP_473375.2 (SEQ ID NO: 78)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpvsi pprnlgy
    Fibronectin 1, isoform 8 precursor, NP_001293058.1 (SEQ ID NO: 79)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsysk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipevpql tdlsfvditd ssiglrwtpl nsstiigyri tvvaagegip ifedfvdssv
    1321 gyytvtglep gidydisvit linggesapt tltqqtavpp ptdlrftnig pdtmrvtwap
    1381 ppsidltnfl vryspvknee dvaelsisps dnavvltnll pgteyvvsys svyeqhestp
    1441 lrgrqktgld sptgidfsdi tansftvhwi apratitgyr irhhpehfsg rpredrvphs
    1501 rnsitltnlt pgteyvvsiv alngreespl ligqqstvsd vprdlevvaa tptslliswd
    1561 apavtvryyr itygetggns pvqeftvpgs kstatisglk pgvdytitvy avtgrgdspa
    1621 sskpisinyr teidkpsqmq vtdvqdnsis vkwlpssspv tgyrvtttpk ngpgptktkt
    1681 agpdqtemti eglqptveyv vsvyaqnpsg esqplvqtav tnidrpkgla ftdvdvdsik
    1741 iawespqgqv sryrvtyssp edgihelfpa pdgeedtael qglrpgseyt vsvvalhddm
    1801 esqpligtqs taipaptdlk ftqvtptsls aqwtppnvql tgyrvrvtpk ektgpmkein
    1861 lapdsssvvv sglmvatkye vsvyalkdtl tsrpaqgvvt tlenvspprr arvtdatett
    1921 itiswrtkte titgfqvdav pangqtpiqr tikpdvrsyt itglqpgtdy kiylytlndn
    1981 arsspvvida staidapsnl rflattpnsl lvswqpprar itgyiikyek pgspprevvp
    2041 rprpgvteat itglepgtey tiyvialknn qksepligrk ktdelpqlvt lphpnlhgpe
    2101 ildvpstvqk tpfvthpgyd tgngiqlpgt sgqqpsvgqq mifeehgfrr ttppttatpi
    2161 rhrprpyppn vgqealsqtt iswapfqdts eyiischpvg tdeeplqfrv pgtstsatlt
    2221 gltrgatyni ivealkdqqr hkvreevvtv gnsvneglnq ptddscfdpy tvshyavgde
    2281 wermsesgfk llcqclgfgs ghfrcdssrw chdngvnyki gekwdrqgen gqmmsctclg
    2341 ngkgefkcdp heatcyddgk tyhvgeqwqk eylgaicsct cfggqrgwrc dncrrpggep
    2401 spegttgqsy nqysciryhqr tntnvncpie cfmpldvqad redsre
    Fibronectin 1, isoform 9 precursor, NP_001293059.1 (SEQ ID NO: 80)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsysk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipevpql tdlsfvditd ssiglrwtpl nsstiigyri tvvaagegip ifedfvdssv
    1321 gyytvtglep gidydisvit linggesapt tltqqtavpp ptdlrftnig pdtmrvtwap
    1381 ppsidltnfl vryspvknee dvaelsisps dnavvltnll pgteyvvsvs svyeqhestp
    1441 lrgrqktgld sptgidfsdi tansftvhwi apratitgyr irhhpehfsg rpredrvphs
    1501 rnsitltnit pgteyvvsiv alngreespl ligqqstvsd vprdlevvaa tptslliswd
    1561 apavtvryyr itygetggns pvqeftvpgs kstatisglk pgvdytitvy avtgrgdspa
    1621 sskpisinyr teidkpsqmq vtdvqdnsis vkwlpssspv tgyrvtttpk ngpgptktkt
    1681 agpdqtemti eglqptveyv vsvyagnpsg esqplvqtav ttipaptdlk ftqvtptsls
    1741 aqwtppnvql tgyrvrvtpk ektgpmkein lapdsssvvv sglmvatkye vsvyalkdtl
    1801 tsrpaqgvvt tlenvspprr arvtdatett itiswrtkte titgfqvdav pangqtpiqr
    1861 tikpdvrsyt itglqpgtdy kiylytlndn arsspvvida staidapsnl rflattpnsl
    1921 lvswqpprar itgyiikyek pgspprevvp rprpgvteat itglepgtey tiyvialknn
    1981 qksepligrk ktgqealsqt tiswapfqdt seyiischpv gtdeeplqfr vpgtstsatl
    2041 tgltrgatyn iivealkdqq rhkvreevvt vgnsvnegln qptddscfdp ytvshyavgd
    2101 ewermsesgf kllcqclgfg sghfrcdssr wchdngvnyk igekwdrqge ngqmmsctcl
    2161 gngkgefkcd pheatcyddg ktyhvgeqwq keylgaicsc tcfggqrgwr cdncrrpgge
    2221 pspegttgqs ynqysqryhq rtntnvncpi ecfmpldvqa dredsre
    Fibronectin 1, isoform 10 precursor, NP_001293060.1 (SEQ ID NO: 81)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsysk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpgveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd
    1321 navvltnllp gteyvvsyss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia
    1381 pratitgyri rhhpehfsgr predrvphsr nsitltnitp gteyvvsiva lngreespll
    1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk
    1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv
    1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge
    1621 sqplvqtavt tipaptdlkf tqvtptslsa qwtppnvqlt gyrvrvtpke ktgpmkeinl
    1681 apdsssvvvs glmvatkyev svyalkdtlt srpaqgvvtt lenvspprra rvtdatetti
    1741 tiswrtktet itgfqvdavp angqtpiqrt ikpdvrsyti tglqpgtdyk iylytlndna
    1801 rsspvvidas taidapsnlr flattpnsll vswqpprari tgyiikyekp gspprevvpr
    1861 prpgvteati tglepgteyt iyvialknnq ksepligrkk tdelpqlvtl phpnlhgpei
    1921 ldvpstvqkt pfvthpgydt gngiqlpgts gqqpsvgqqm ifeehgfrrt tppttatpir
    1981 hrprpyppnv gqealsqtti swapfqdtse yiischpvgt deeplqfrvp gtstsatltg
    2041 ltrgatynii vealkdqqrh kvreevvtvg nsvneglnqp tddscfdpyt vshyavgdew
    2101 ermsesgfkl lcqclgfgsg hfrcdssrwc hdngvnykig ekwdrqgeng qmmsctclgn
    2161 gkgefkcdph eatcyddgkt yhvgeqwqke ylgaicsctc fggqrgwrcd ncrrpggeps
    2221 pegttgqsyn gysqryhqrt ntnvncpiec fmpldvqadr edsre
    Fibronectin 1, isoform 11 precursor, NP_001293061.1 (SEQ ID NO: 82)
    1 mlrgpgpgll llavqclgta vpstgasksk rqaqqmvqpq spvaysqskp gcydngkhyq
    61 inqqwertyl gnalvctcyg gsrgfncesk peaeetcfdk ytgntyrvgd tyerpkdsmi
    121 wdctcigagr grisctianr cheggqsyki gdtwrrphet ggymlecvcl gngkgewtck
    181 piaekcfdha agtsyvvget wekpyqgwmm vdctclgegs gritctsrnr cndqdtrtsy
    241 rigdtwskkd nrgnllqcic tgngrgewkc erhtsvqtts sgsgpftdvr aavyqpqphp
    301 qpppyghcvt dsgvvysvgm qwlktqgnkq mlctclgngv scqetavtqt yggnsngepc
    361 vlpftyngrt fyscttegrq dghlwcstts nyeqdqkysf ctdhtvlvqt rggnsngalc
    421 hfpflynnhn ytdctsegrr dnmkwcgttq nydadqkfgf cpmaaheeic ttnegvmyri
    481 gdqwdkqhdm ghmmrctcvg ngrgewtcia ysqlrdqciv dditynvndt fhkrheeghm
    541 lnctcfgqgr grwkcdpvdq cqdsetgtfy qigdswekyv hgvryqcycy grgigewhcq
    601 plqtypsssg pvevfitetp sqpnshpiqw napqpshisk yilrwrpkns vgrwkeatip
    661 ghlnsytikg lkpgvvyegq lisiqqyghq evtrfdfttt ststpvtsnt vtgettpfsp
    721 lvatsesvte itassfvvsw vsasdtvsgf rveyelseeg depqyldlps tatsvnipdl
    781 lpgrkyivnv yqisedgeqs lilstsqtta pdappdptvd qvddtsivvr wsrpqapitg
    841 yrivyspsve gsstelnlpe tansvtlsdl qpgvqyniti yaveenqest pvviqqettg
    901 tprsdtvpsp rdlqfvevtd vkvtimwtpp esavtgyrvd vipvnlpgeh gqrlpisrnt
    961 faevtglspg vtyyfkvfav shgreskplt aqqttkldap tnlqfvnetd stvlvrwtpp
    1021 raqitgyrlt vgltrrgqpr qynvgpsysk yplrnlqpas eytvslvaik gnqespkatg
    1081 vfttlqpgss ippyntevte ttivitwtpa prigfklgvr psqggeapre vtsdsgsivv
    1141 sgltpqveyv ytiqvlrdgq erdapivnkv vtplspptnl hleanpdtgv ltvswerstt
    1201 pditgyritt tptngqqgns leevvhadqs sctfdnlspg leynvsvytv kddkesvpis
    1261 dtiipavppp tdlrftnigp dtmrvtwapp psidltnflv ryspvkneed vaelsispsd
    1321 navvltnllp gteyvvsvss vyeqhestpl rgrqktglds ptgidfsdit ansftvhwia
    1381 pratitgyri rhhpehfsgr predrvphsr nsitltnltp gteyvvsiva lngreespll
    1441 igqqstvsdv prdlevvaat ptslliswda pavtvryyri tygetggnsp vqeftvpgsk
    1501 statisglkp gvdytitvya vtgrgdspas skpisinyrt eidkpsqmqv tdvqdnsisv
    1561 kwlpssspvt gyrvtttpkn gpgptktkta gpdqtemtie glqptveyvv svyaqnpsge
    1621 sqplvqtavt tipaptdlkf tqvtptslsa qwtppnvqlt gyrvrvtpke ktgpmkeinl
    1681 apdsssvvvs glmvatkyev svyalkdtlt srpaqgvvtt lenvspprra rvtdatetti
    1741 tiswrtktet itgfqvdavp angqtpiqrt ikpdvrsyti tglqpgtdyk iylytlndna
    1801 rsspvvidas taidapsnlr flattpnsll vswqpprari tgyiikyekp gspprevvpr
    1861 prpgvteati tglepgteyt iyvialknnq ksepligrkk tvqktpfvth pgydtgngiq
    1921 lpgtsgqqps vgqqmifeeh gfrrttpptt atpirhrprp yppnvgqeal sqttiswapf
    1981 qdtseyiisc hpvgtdeepl qfrvpgtsts atltgltrga tyniivealk dqqrhkvree
    2041 vvtvgnsvne glnqptddsc fdpytvshya vgdewermse sgfkllcqcl gfgsghfrcd
    2101 ssrwchdngv nykigekwdr qgengqmmsc tclgngkgef kcdpheatcy ddgktyhvge
    2161 qwgkeylgai csctcfggqr gwrcdncrrp ggepspegtt gqsynqysqr yhqrtntnvn
    2221 cpiecfmpld vqadredsre
    Major histocompatibility complex, class II, DR beta 1, precursor,
    NP_001230894.1 (SEQ ID NO: 83)
    1 mvclrlpggs cmavltvtlm vlssplalag dtrprfleys tsechffngt ervryldrvf
    61 hnqeenvrfd sdvgefravt elgrpdaeyw nsqkdlleqk rgrvdnycrh nygvvesftv
    121 qrrvhpkvtv ypsktqplqh hnllvcsysg fypgsievrw frngqeektg vvstglihng
    181 dwtfqtlvml etvprsgevy tcqvehpsvt spltvewrar sesaqskmls gvggfvlgll
    241 flgaglfiyf rnqkghsglq prgfls
    Major histocompatibility complex, class II, DR beta 1, precursor,
    NP_001346122.1 (SEQ ID NO: 84)
    1 mvclklpggs cmaaltvtlm vlssplalag dtqprflwqg kykchffngt ervqflerlf
    61 ynqeefvrfd sdvgeyravt elgrpvaesw nsqkdiledr rgqvdtvcrh nygvgesftv
    121 qrrvhpevtv ypaktqplqh hnllvcsysg fypgsievrw frngqeekag vvstgliqng
    181 dwtfqtlvml etvprsgevy tcqvehpsvm spltvewrar sesaqskmls gvggfvlgll
    241 flgaglfiyf rnqkghsglq ptgfls
    Major histocompatibility complex, class II, DR beta 1, precursor,
    NP_001346123.1 (SEQ ID NO: 85)
    1 mvclkfpggs cmaaltvtlm vlssplalag dtrprfleqv khechffngt ervrfldrvf
    61 yhqeeyvrfd sdvgeyravt elgrpdaeyw nsqkdlleqr raevdtycrh nygvvesftv
    121 qrrvypevtv ypaktqplqh hnllvcsvng fypgsievrw frngqeektg vvstgliqng
    181 dwtfqtlvml etvprsgevy tcqvehpslt spltvewrar sesaqskmls gvggfvlgll
    241 flgaglfiyf rnqkghsglq ptgfls
    Major histocompatibility complex, class II, DR beta 1, precursor, NP_002115.2
    (SEQ ID NO: 86)
    1 mvclklpggs cmtaltvtlm vlssplalsg dtrprflwqp krechffngt ervrfldrvf
    61 ynqeesvrfd sdvgefravt elgrpdaeyw nsqkdileqa raavdtycrh nygvvesftv
    121 qrrvqpkvtv ypsktqplqh hnllvcsysg fypgsievrw flngqeekag mvstgliqng
    181 dwtfqtlvml etvprsgevy tcqvehpsvt spltvewrar sesaqskmls gvggfvlgll
    241 flgaglfiyf rnqkghsglq ptgfls
    Major histocompatibility complex, class II, DR beta 5, precursor, NP_002116.2
    (SEQ ID NO: 87)
    1 mvclklpggs ymakltvtlm vlssplalag dtrprflqqd kyechffngt ervrflhrdi
    61 ynqeedlrfd sdvgeyravt elgrpdaeyw nsqkdfledr raavdtycrh nygvgesftv
    121 qrrvepkvtv ypartqtlqh hnllvcsvng fypgsievrw frnsqeekag vvstgliqng
    181 dwtfqtlvml etvprsgevy tcqvehpsvt spltvewraq sesaqskmls gvggfvlgll
    241 flgaglfiyf knqkghsglh ptglvs
    Hydroxysteroid 17-beta dehydrogenase 3, NP_000188.1 (SEQ ID NO: 88)
    1 mgdvleqffi ltgllvclac lakcvrfsrc vllnywkvlp ksflrsmgqw avitgagdgi
    61 gkaysfelak rglnvvlisr tlekleaiat eierttgrsv kiiqadftkd diyehikekl
    121 agleigilvn nvgmlpnllp shflnapdei qslihcnits vvkmtqlilk hmesrqkgli
    181 lnissgialf pwplysmysa skafvcafsk alqeeykake viiqvltpya vstamtkyln
    241 tnvitktade fvkeslnyvt iggetcgcla heilagflsl ipawafysga fqrlllthyv
    301 aylklntkvr
    Insulin degrading enzyme, isoform 1, NP_004960.2 (SEQ ID NO: 89)
    1 mryrlawllh palpstfrsv lgarlppper lcgfqkktys kmnnpaikri gnhitksped
    61 kreyrglela ngikvllisd pttdkssaal dvhigslsdp pniaglshfc ehmlflgtkk
    121 ypkeneysqf lsehagssna ftsgehtnyy fdvshehleg aldrfaqffl cplfdesckd
    181 revnavdseh eknvmndawr lfqlekatgn pkhpfskfgt gnkytletrp nqegidvrqe
    241 llkfhsayys snlmavcvlg reslddltnl vvklfseven knvplpefpe hpfqeehlkq
    301 lykivpikdi rnlyvtfpip dlqkyyksnp ghylghligh egpgsllsel kskgwvntlv
    361 ggqkegargf mffiinvdlt eegllhvedi ilhmfqyiqk lraegpqewv fqeckdlnav
    421 afrfkdkerp rgytskiagi lhyypleevl taeylleefr pdliemvldk lrpenvrvai
    481 vsksfegktd rteewygtqy kqeaipdevi kkwqnadlng kfklptknef iptnfeilpl
    541 ekeatpypal ikdtamsklw fkqddkfflp kaclnfeffs pfayvdplhc nmaylylell
    601 kdslneyaya aelaglsydl qntiygmyls vkgyndkqpi llkkiiekma tfeidekrfe
    661 iikeaymrsl nnfraeqphq hamyylrllm tevawtkdel kealddvtlp rlkafipqll
    721 srlhieallh gnitkqaalg imqmvedtli ehahtkpllp sqlvryrevq lpdrgwfvyq
    781 qrnevhnncg ieiyyqtdmq stsenmflel fcqiisepcf ntlrtkeqlg yivfsgprra
    841 ngiqglrfii qsekpphyle srveaflitm eksiedmtee afqkhiqala irrldkpkkl
    901 saecakywge iisqqynfdr dntevaylkt ltkediikfy kemlavdapr rhkvsvhvla
    961 remdscpvvg efpcqndinl sqapalpqpe viqnmtefkr glplfplvkp hinfmaakl
    Insulin degrading enzyme, isoform 2, NP_001159418.1 (SEQ ID NO: 90)
    1 msklwfkqdd kfflpkacln feffspfayv dplhcnmayl ylellkdsln eyayaaelag
    61 lsydlqntiy gmylsvkgyn dkqpillkki iekmatfeid ekrfeiikea ymrslnnfra
    121 eqphqhamyy lrllmtevaw tkdelkeald dvtlprlkaf ipqllsrlhi eallhgnitk
    181 qaalgimqmv edtliehaht kpllpsqlvr yrevqlpdrg wfvyqqrnev hnncgieiyy
    241 qtdmqstsen mflelfcqii sepcfntlrt keqlgyivfs gprrangiqg lrfiiqsekp
    301 phylesrvea flitmeksie dmteeafqkh iqalairrld kpkklsaeca kywgeiisqq
    361 ynfdrdntev aylktltked iikfykemla vdaprrhkvs vhvlaremds cpvvgefpcq
    421 ndinlsqapa lpqpeviqnm tefkrglplf plvkphinfm aakl
    Insulin degrading enzyme, isoform 3, NP_001309722.1 (SEQ ID NO: 91)
    1 mryrlawllh palpstfrsv lgarlppper lcgfqkktys kmnnpaikri gnhitksped
    61 kreyrglela ngikvllisd pttdkssaal dvhigslsdp pniaglshfc ehmlflgtkk
    121 ypkeneysqf lsehagssna ftsgehtnyy fdvshehleg aldrfaqffl cplfdesckd
    181 revnavdseh eknvmndawr lfqlekatgn pkhpfskfgt gnkytletrp nqegidvrqe
    241 llkfhsayys snlmavcvlg reslddltnl vvklfseven knvplpefpe hpfqeehlkq
    301 lykivpikdi rnlyvtfpip dlqkyyksnp ghylghligh egpgsllsel kskgwvntlv
    361 ggqkegargf mffiinvdlt eegllhvedi ilhmfqyiqk lraegpqewv fqeckdlnav
    421 afrfkdkerp rgytskiagi lhyypleevl taeylleefr pdliemvldk lrpenvrvai
    481 vsksfegktd rteewygtqy kqeaipdevi kkwqnadlng kfklptknef iptnfeilpl
    541 ekeatpypal ikdtamsklw fkqddkfflp kaclnfeffs ryiyadplhc nmtylfirll
    601 kddlkeytya arlsglsygi asgmnaills vkgyndkqpi llkkiiekma tfeidekrfe
    661 iikeaymrsl nnfraeqphq hamyylrllm tevawtkdel kealddvtlp rlkafipqll
    721 srlhieallh gnitkqaalg imqmvedtli ehahtkpllp sqlvryrevq lpdrgwfvyq
    781 qrnevhnncg ieiyyqtdmq stsenmflel fcqiisepcf ntlrtkeqlg yivfsgprra
    841 ngiqglrfii qsekpphyle srveaflitm eksiedmtee afqkhigala irrldkpkkl
    901 saecakywge iisqqynfdr dntevaylkt ltkediikfy kemlavdapr rhkvsvhvla
    961 remdscpvvg efpcqndinl sqapalpqpe viqnmtefkr glplfplvkp hinfmaakl
    Insulin degrading enzyme, isoform 4, NP_001309723.1 (SEQ ID NO: 92)
    1 mryrlawllh palpstfrsv lgarlppper lcgfqkktys kmnnpaikri gnhitksped
    61 kreyrglela ngikvllisd pttdkssaal dvhigslsdp pniaglshfc ehmlflgtkk
    121 ypkeneysqf lsehagssna ftsgehtnyy fdvshehleg aldrfaqffl cplfdesckd
    181 revnavdseh eknvmndawr lfqlekatgn pkhpfskfgt greslddltn lvvklfseve
    241 nknvplpefp ehpfqeehlk qlykivpikd irnlyvtfpi pdlqkyyksn pghylghlig
    301 hegpgsllse lkskgwvntl vggqkegarg fmffiinvdl teegllhved iilhmfqyiq
    361 klraegpqew vfqeckdlna vafrfkdker prgytskiag ilhyypleev ltaeylleef
    421 rpdliemvld klrpenvrva ivsksfegkt drteewygtq ykqeaipdev ikkwqnadln
    481 gkfklptkne fiptnfeilp lekeatpypa likdtamskl wfkqddkffl pkaclnfeff
    541 spfayvdplh cnmaylylel lkdslneyay aaelaglsyd lqntiygmyl svkgyndkqp
    601 illkkiiekm atfeidekrf eiikeaymrs lnnfraeqph qhamyylrll mtevawtkde
    661 lkealddvtl prlkafipql lsrlhieall hgnitkqaal gimqmvedtl iehahtkpll
    721 psqlvryrev qlpdrgwfvy qqrnevhnnc gieiyyqtdm qstsenmfle lfcqiisepc
    781 fntlrtkeql gyivfsgprr angiqglrfi iqsekpphyl esrveaflit meksiedmte
    841 eafqkhiqal airrldkpkk lsaecakywg eiisqqynfd rdntevaylk tltkediikf
    901 ykemlavdap rrhkvsvhvl aremdscpvv gefpcqndin lsqapalpqp eviqnmtefk
    961 rglplfplvk phinfmaakl
    Insulin degrading enzyme, isoform 5, NP_001309724.1, NP_001309725.1 (SEQ ID
    NO: 93)
    1 mnnpaikrig nhitkspedk reyrglelan gikvllisdp ttdkssaald vhigslsdpp
    61 niaglshfce hmlflgtkky pkeneysqfl sehagssnaf tsgehtnyyf dvshehlega
    121 ldrfaqfflc plfdesckdr evnavdsehe knvmndawrl fqlekatgnp khpfskfgtg
    181 nkytletrpn qegidvrqel lkfhsayyss nlmavcvlgr eslddltnlv vklfsevenk
    241 nvplpefpeh pfqeehlkql ykivpikdir nlyvtfpipd lqkyyksnpg hylghlighe
    301 gpgsllselk skgwvntlvg gqkegargfm ffiinvdlte egllhvedii lhmfqyiqkl
    361 raegpgewvf qeckdlnava frfkdkerpr gytskiagil hyypleevlt aeylleefrp
    421 dliemvldkl rpenvrvaiv sksfegktdr teewygtqyk qeaipdevik kwqnadlngk
    481 fklptknefi ptnfeilple keatpypali kdtamsklwf kqddkfflpk aclnfeffsp
    541 fayvdplhcn maylylellk dslneyayaa elaglsydlq ntiygmylsv kgyndkqpil
    601 lkkiiekmat feidekrfei ikeaymrsln nfraeqphqh amyylrllmt evawtkdelk
    661 ealddvtlpr lkafipqlls rlhieallhg nitkqaalgi mqmvedtlie hahtkpllps
    721 qlvryrevql pdrgwfvyqq rnevhnncgi eiyyqtdmqs tsenmflelf cqiisepcfn
    781 tlrtkeqlgy ivfsgprran giqglrfiiq sekpphyles rveaflitme ksiedmteea
    841 fqkhiqalai rrldkpkkls aecakywgei isqqynfdrd ntevaylktl tkediikfyk
    901 emlavdaprr hkvsvhvlar emdscpvvge fpcqndinls qapalpqpev iqnmtefkrg
    961 lplfplvkph infmaakl
    Insulin degrading enzyme, isoform 6, NP_001309726.1 (SEQ ID NO: 94)
    1 msklwfkqdd kfflpkacln feffsryiya dplhcnmtyl firllkddlk eytyaarlsg
    61 lsygiasgmn aillsvkgyn dkqpillkki iekmatfeid ekrfeiikea ymrslnnfra
    121 eqphqhamyy lrllmtevaw tkdelkeald dvtlprlkaf ipqllsrlhi eallhgnitk
    181 qaalgimqmv edtliehaht kpllpsqlvr yrevqlpdrg wfvyqqrnev hnncgieiyy
    241 qtdmqstsen mflelfcqii sepcfntlrt keqlgyivfs gprrangiqg lrfiiqsekp
    301 phylesrvea flitmeksie dmteeafqkh iqalairrld kpkklsaeca kywgeiisqq
    361 ynfdrdntev aylktltked iikfykemla vdaprrhkvs vhvlaremds cpvvgefpcq
    421 ndinlsqapa lpqpeviqnm tefkrglplf plvkphinfm aakl
    Indoleamine 2,3-dioxygenase 1, NP_002155.1 (SEQ ID NO: 95)
    1 mahamenswt iskeyhidee vgfalpnpqe nlpdfyndwm fiakhlpdli esgqlrerve
    61 klnmlsidhl tdhksqrlar lvlgcitmay vwgkghgdvr kvlprniavp ycqlskklel
    121 ppilvyadcv lanwkkkdpn kpltyenmdv lfsfrdgdcs kgfflvsllv eiaaasaikv
    181 iptvfkamqm qerdtllkal leiascleka lqvfhqihdh vnpkaffsvl riylsgwkgn
    241 pqlsdglvye gfwedpkefa ggsagqssvf qcfdvllgiq qtaggghaaq flqdmrrymp
    301 pahrnflcsl esnpsvrefv lskgdaglre aydacvkalv slrsyhlqiv tkyilipasq
    361 qpkenktsed pskleakgtg gtdlmnflkt vrstteksll keg
    Insulin like growth factor binding protein 5, precursor, NP_000590.1 (SEQ ID
    NO: 96)
    1 mvlltavlll laayagpaqs lgsfvhcepc dekalsmcpp splgcelvke pgcgccmtca
    61 laegqscgvy tercaqglrc lprqdeekpl hallhgrgvc lneksyreqv kierdsrehe
    121 epttsemaee tyspkifrpk htriselkae avkkdrrkkl tqskfvggae ntahpriisa
    181 pemrqeseqg pcrrhmeasl qelkasprmv pravylpncd rkgfykrkqc kpsrgrkrgi
    241 cwcvdkygmk lpgmeyvdgd fqchtfdssn ve
    Insulin like growth factor binding protein 7, isoform 1 precursor,
    NP_001544.1 (SEQ ID NO: 97)
    1 merpslrall lgaaglllll lplssssssd tcgpcepasc pplpplgcll getrdacgcc
    61 pmcargegep cggggagrgy capgmecvks rkrrkgkaga aaggpgvsgv cvcksrypvc
    121 gsdgttypsg cqlraasqra esrgekaitq vskgtceqgp sivtppkdiw nvtgaqvyls
    181 cevigiptpv liwnkvkrgh ygvqrtellp gdrdnlaiqt rggpekhevt gwvlvsplsk
    241 edageyecha snsqggasas akitvvdalh eipvkkgega el
    Insulin like growth factor binding protein 7, isoform 2 precursor,
    NP_001240764.1 (SEQ ID NO: 98)
    1 merpslrall lgaaglllll lplssssssd tcgpcepasc pplpplgcll getrdacgcc
    61 pmcargegep cggggagrgy capgmecvks rkrrkgkaga aaggpgvsgv cvcksrypvc
    121 gsdgttypsg cqlraasqra esrgekaitq vskgtceqgp sivtppkdiw nvtgaqvyls
    181 cevigiptpv liwnkvkrgh ygvqrtellp gdrdnlaiqt rggpekhevt gwvlvsplsk
    241 edageyecha snsqgqasas akitvvdalh eipvkkgtq
    Potassium two pore domain channel subfamily K member 1, NP_002236.1 (SEQ ID
    NO: 99)
    1 mlqslagssc vrlverhrsa wcfgflvlgy llylvfgavv fssvelpyed llrqelrklk
    61 rrfleehecl seqqleqflg rvleasnygv svlsnasgnw nwdftsalff astvlsttgy
    121 ghtvplsdgg kafciiysvi gipftllflt avvqritvhv trrpvlyfhi rwgfskqvva
    181 ivhavllgfv tvscfffipa avfsvleddw nflesfyfcf islstiglgd yvpgegynqk
    241 frelykigit cylllgliam lvvletfcel helkkfrkmf yvkkdkdedq vhiiehdqls
    301 fssitdqaag mkedqkqnep fvatqssacv dgpanh
    Lysosomal associated membrane protein 3, precursor, NP_055213.2 (SEQ ID NO:
    100)
    1 mprqlsaaaa lfaslavilh dgsqmrakaf petrdysqpt aaatvqdikk pvqqpakqap
    61 hqtlaarfmd ghitfqtaat vkiptttpat tkntattspi tytlvttqat pnnshtappv
    121 tevtvgpsla pyslpptitp pahttgtsss tvshttgntt gpsnqttlpa tlsialhkst
    181 tgqkpvqpth apgttaaahn ttrtaapast vpgptlapqp ssvktgiyqv lngsrlcika
    241 emgiqlivqd kesvfsprry fnidpnatqa sgncgtrksn lllnfqggfv nltftkdees
    301 yyisevgayl tvsdpetiyq gikhavvmfq tavghsfkcv seqslqlsah lqvkttdvql
    361 qafdfeddhf gnvdecssdy tivlpvigai vvglclmgmg vykirlrcqs sgyqri
    MAGE family member B2, NP_002355.2 (SEQ ID NO: 101)
    1 mprgqksklr arekrrkard etrglnvpqv teaeeeeapc csssysggaa ssspaagipq
    61 epqrapttaa aaaagvsstk skkgakshqg eknasssqas tstkspsedp ltrksgslvq
    121 fllykykikk svtkgemlki vgkrfrehfp eilkkasegl svvfglelnk vnpnghtytf
    181 idkvdltdee sllsswdfpr rkllmpllgv iflngnsate eeiweflnml gvydgeehsv
    241 fgepwklitk dlvqekyley kqvpssdppr fqflwgpray aetskmkvle flakvngttp
    301 cafpthyeea lkdeekagv
    Mitogen-activated protein kinase 13, NP_002745.1 (SEQ ID NO: 102)
    1 mslirkkgfy kqdvnktawe lpktyvspth vgsgaygsvc saidkrsgek vaikklsrpf
    61 qseifakray rellllkhmq henviglldv ftpasslrnf ydfylvmpfm qtdlqkimgm
    121 efseekigyl vyqmlkglky ihsagvvhrd lkpgnlavne dcelkildfg larhadaemt
    181 gyvvtrwyra pevilswmhy nqtvdiwsvg cimaemltgk tlfkgkdyld qltqilkvtg
    241 vpgtefvqkl ndkaaksyiq slpqtprkdf tqlfpraspq aadllekmle ldvdkrltaa
    301 qalthpffep frdpeeetea qqpfddsleh ekltvdewkq hiykeivnfs piarkdsrrr
    361 sgmkl
    Macrophage receptor with collagenous structure, NP_006761.1 (SEQ ID NO: 103)
    1 mrnkkilked ellsetqqaa fhqiamepfe invpkpkrrn gvnfslavvv iylilltaga
    61 gllvvqvlnl qarlrvlemy flndtlaaed spsfsllqsa hpgehlaqga srlqvlqaql
    121 twvrvshehl lqrvdnftqn pgmfrikgeq gapglqghkg amgmpgapgp pgppaekgak
    181 gamgrdgatg psgpqgppgv kgeaglqgpq gapgkqgatg tpgpqgekgs kgdggligpk
    241 getgtkgekg dlglpgskgd rgmkgdagvm gppgaqgskg dfgrpgppgl agfpgakgdg
    301 gqpglqgvpg ppgavghpga kgepgsagsp graglpgspg spgatglkgs kgdtglqgqq
    361 grkgesgvpg pagvkgeqgs pglagpkgap gqagqkgdqg vkgssgeqgv kgekgergen
    421 sysvrivgss nrgraevyys gtwgticdde wqnsdaivfc rmlgyskgra lykvgagtgq
    481 iwldnvqcrg testlwsctk nswghhdcsh eedagvecsv
    Malic enzyme 1, NADP-dependent malic enzyme, NP_002386.1 (SEQ ID NO: 104)
    1 mepeaprrrh thqrgylltr nphlnkdlaf tleerqqlni hgllppsfns qeiqvlrvvk
    61 nfehlnsdfd rylllmdlqd rneklfyrvl tsdiekfmpi vytptvglac qqyslvfrkp
    121 rglfitihdr ghiasvinaw pedvikaivv tdgerilglg dlgcngmgip vgklalytac
    181 ggmnpqeclp vildvgtene ellkdplyig lrqrrvrgse yddfldefme aysskygmnc
    241 liqfedfanv nafrllnkyr nqyctfnddi qgtasvavag llaalritkn klsdqtilfq
    301 gageaalgia hlivmaleke glpkekaikk iwlvdskgli vkgrasltqe kekfahehee
    361 mknleaivqe ikptaligva aiggafseqi lkdmaafner piifalsnpt skaecsaeqc
    421 ykitkgraif asgspfdpvt lpngqtlypg qgnnsyvfpg valgvvacgl rqitdniflt
    481 taeviaqqvs dkhleegrly pplntirdvs lkiaekivkd ayqektatvy pepqnkeafv
    541 rsqmystdyd qilpdcyswp eevqkiqtkv dq
    Migration and invasion inhibitory protein, NP_068752.2 (SEQ ID NO: 105)
    1 mveaeelaql rllnlellrq lwvgqdavrr svaraasess lessssynse tpstpetsst
    61 slstscprgr ssvwgppdac rgdlrdvars gvaslppakc qhqeslgrpr phsapslgts
    121 slrdpepsgr lgdpgpqeaq tprsilaqqs klskprvtfs eesavpkrsw rlrpylgydw
    181 iagsldtsss itsqpeaffs klqefretnk eecicshpep qlpglressg sgveedhecv
    241 ycyrvnrrlf pvpvdpgtpc rlcrtprdqq gpgtlaqpah vrvsiplsil epphryhihr
    301 rksfdasdtl alprhcllgw difppkseks saprnldlws sysaeaqhqk lsgtsspfhp
    361 aspmqmlppt ptwsvpqvpr phvprqkp
    Matrix metallopeptidase 12, macrophage metalloelastase preproprotein,
    NP_002417.2 (SEQ ID NO: 106)
    1 mkfllilllq atasgalpln sstsleknnv lfgerylekf ygleinklpv tkmkysgnlm
    61 kekiqemqhf lglkvtgqld tstlemmhap rcgvpdvhhf rempggpvwr khyityrinn
    121 ytpdmnredv dyairkafqv wsnvtplkfs kintgmadil vvfargahgd fhafdgkggi
    181 lahafgpgsg iggdahfded efwtthsggt nlfltavhei ghslglghss dpkavmfpty
    241 kyvdintfrl saddirgiqs lygdpkenqr lpnpdnsepa lcdpnlsfda vttvgnkiff
    301 fkdrffwlkv serpktsvnl isslwptlps gieaayeiea rnqvflfkdd kywlisnlrp
    361 epnypksihs fgfpnfvkki daavfnprfy rtyffvdnqy wryderrqmm dpgypklitk
    421 nfqgigpkid avfysknkyy yffqgsnqfe ydfllqritk tlksnswfgc
    Matrix metallopeptidase 7, matrilysin preproprotein, NP_002414.1 (SEQ ID NO:
    107)
    1 mrltvlcavc llpgslalpl pqeaggmsel qweqaqdylk rfylydsetk nansleaklk
    61 emqkffglpi tgmlnsrvie imqkprcgvp dvaeyslfpn spkwtskvvt yrivsytrdl
    121 phitvdrlvs kalnmwgkei plhfrkvvwg tadimigfar gahgdsypfd gpgntlahaf
    181 apgtglggda hfdederwtd gsslginfly aathelghsl gmghssdpna vmyptygngd
    241 pqnfklsqdd ikgigklygk rsnsrkk
    Myelin protein zero like 1, myelin protein zero-like protein 1 isoform a
    precursor, NP_003944.1 (SEQ ID NO: 108)
    1 maasagagav iaapdsrrwl wsvlaaalgl ltagvsalev ytpkeifvan gtqgkltckf
    61 kststtgglt syswsfqpeg adttvsffhy sqgqvylgny ppfkdriswa gdldkkdasi
    121 nienmqfihn gtyicdvknp pdivvqpghi rlyvvekenl pvfpvwvvvg ivtavvlglt
    181 llismilavl yrrknskrdy tgcstsesls pvkqaprksp sdteglvksl psgshqgpvi
    241 yaqldhsggh hsdkinkses vvyadirkn
    Myelin protein zero like 1, myelin protein zero-like protein 1 isoform b
    precursor, NP_078845.3 (SEQ ID NO: 109)
    1 maasagagav iaapdsrrwl wsvlaaalgl ltagvsalev ytpkeifvan gtqgkltckf
    61 kststtgglt syswsfqpeg adttvsffhy sqgqvylgny ppfkdriswa gdldkkdasi
    121 nienmqfihn gtyicdvknp pdivvqpghi rlyvvekenl pvfpvwvvvg ivtavvlglt
    181 llismilavl yrrknskrdy tgaqsymhs
    Myelin protein zero like 1, myelin protein zero-like protein 1 isoform c
    precursor, NP_001139663.1 (SEQ ID NO: 110)
    1 maasagagav iaapdsrrwl wsvlaaalgl ltagvsalev ytpkeifvan gtqgkltckf
    61 kststtgglt syswsfqpeg adttvsgpvi yaqldhsggh hsdkinkses vvyadirkn
    Macrophage scavenger receptor 1, macrophage scavenger receptor types I and II
    isoform type 1, NP_619729.1 (SEQ ID NO: lll)
    1 meqwdhfhnq qedtdscses vkfdarsmta llppnpknsp slqeklksfk aalialyllv
    61 favlipligi vaaqllkwet kncsvsstna nditqsltgk gndseeemrf qevfmehmsn
    121 mekriqhild meanlmdteh fqnfsmttdq rfndillqls tlfssvqghg naideisksl
    181 islnttlldl qlnienlngk iqentfkqqe eiskleervy nvsaeimamk eeqvhleqei
    241 kgevkvlnni tndlrlkdwe hsqtlrnitl iqgppgppge kgdrgptges gprgfpgpig
    301 ppglkgdrga igfpgsrglp gyagrpgnsg pkgqkgekgs gntltpftkv rlvggsgphe
    361 grveilhsgq wgticddrwe vrvgqvvcrs lgypgvqavh kaahfgqgtg piwlnevfcf
    421 gressieeck irqwgtracs hsedagvtct l
    Macrophage scavenger receptor 1, macrophage scavenger receptor types I and II
    isoform type 2, NP_002436.1 (SEQ ID NO: 112)
    1 meqwdhfhnq qedtdscses vkfdarsmta llppnpknsp slqeklksfk aalialyllv
    61 favlipligi vaaqllkwet kncsysstna nditqsltgk gndseeemrf qevfmehmsn
    121 mekriqhild meanlmdteh fqnfsmttdq rfndillqls tlfssvqghg naideisksl
    181 islnttlldl qlnienlngk iqentfkqqe eiskleervy nvsaeimamk eegvhleqei
    241 kgevkvlnni tndlrlkdwe hsqtlrnitl iqgppgppge kgdrgptges gprgfpgpig
    301 ppglkgdrga igfpgsrglp gyagrpgnsg pkgqkgekgs gntlrpvqlt dhiragps
    Macrophage scavenger receptor 1, macrophage scavenger receptor types I and II
    isoform type 3, NP_619730.1 (SEQ ID NO: 113)
    1 meqwdhfhnq qedtdscses vkfdarsmta llppnpknsp slqeklksfk aalialyllv
    61 favlipligi vaaqllkwet kncsvsstna nditqsltgk gndseeemrf qevfmehmsn
    121 mekriqhild meanlmdteh fqnfsmttdq rfndillqls tlfssvqghg naideisksl
    181 islnttlldl qlnienlngk iqentfkqqe eiskleervy nvsaeimamk eeqvhleqei
    241 kgevkvlnni tndlrlkdwe hsqtlrnitl iqgppgppge kgdrgptges gprgfpgpig
    301 ppglkgdrga igfpgsrglp gyagrpgnsg pkgqkgekgs gntlstgpiw lnevfcfgre
    361 ssieeckirq wgtracshse dagvtctl
    Myoneurin, isoform A, NP_001172047.1, NP_061127.1 (SEQ ID NO: 114)
    1 mqyshhcehl lerlnkqrea gflcdctivi gefqfkahrn vlasfseyfg aiyrstsenn
    61 vfldqsqvka dgfqkllefi ytgtlnldsw nvkeihqaad ylkveevvtk ckikmedfaf
    121 ianpssteis sitgnielnq qtclltlrdy nnreksevst dliqanpkqg alakkssqtk
    181 kkkkafnspk tgqnktvqyp sdilenasve lfldanklpt pvveqvaqin dnseleltsv
    241 ventfpaqdi vhtvtvkrkr gksqpncalk ehsmsniasv kspyeaensg eeldqryska
    301 kpmcntcgkv fseasslrrh mrihkgvkpy vchlcgkaft qcnqlkthvr thtgekpykc
    361 elcdkgfaqk cqlvfhsrmh hgeekpykcd vcnlqfatss nlkiharkhs gekpyvcdrc
    421 gqrfaqastl tyhvrrhtge kpyvcdtcgk afavssslit hsrkhtgekp yicgicgksf
    481 issgelnkhf rshtgerpfi celcgnsytd iknlkkhktk vhsgadktld ssaedhtlse
    541 qdsiqkspls etmdvkpsdm tlplalplgt edhhmllpvt dtqsptsdtl lrstvngyse
    601 pqliflqqly
    Myoneurin, isoform B, NP_001172048.1 (SEQ ID NO: 115)
    1 mqyshhcehl lerlnkqrea gflcdctivi gefqfkahrn vlasfseyfg aiyrstsenn
    61 vfldqsqvka dgfqkllefi ytgtlnldsw nvkeihqaad ylkveevvtk ckikmedfaf
    121 ianpssteis sitgnielnq qtclltlrdy nnreksevst dliqanpkqg alakkssqtk
    181 kkkkafnspk tgqnktvqyp sdilenasve lfldanklpt pvveqvaqin dnseleltsv
    241 ventfpaqdi vhtvtvkrkr gksqpncalk ehsmsniasv kspyeaensg eeldqryska
    301 kpmcntcgkv fseasslrrh mrihkgvkpy vchlcgkaft qcnqlkthvr thtgekpykc
    361 elcdkgfaqk cqlvfhsrmh hgeekpykcd vcnlqfatss nlkiharkhs gekpyvcdrc
    421 gqrfaqastl tyhvrrhtge kpyvcdtcgk afayssslit hsrkhtgekp yicgicgksf
    481 issgelnkhf rshtgadktl dssaedhtls eqdsiqkspl setmdvkpsd mtlplalplg
    541 tedhhmllpv tdtqsptsdt llrstvngys epqliflqql y
    N-acetylglucosamine kinase, isoform 1, NP_060037.3 (SEQ ID NO: 116)
    1 mrtrtgsqla arevtgsgav prqlegrrcq agrdanggts sdgsssmaai yggvegggtr
    61 sevllvsedg kilaeadgls tnhwligtdk cverinemvn rakrkagvdp lvplrslgls
    121 lsggdqedag rilieelrdr fpylsesyli ttdaagsiat atpdggvvli sgtgsncrli
    181 npdgsesgcg gwghmmgdeg saywiahqav kivfdsidnl eaaphdigyv kqamfhyfqv
    241 pdrlgilthl yrdfdkcrfa gfcrkiaega qqgdplsryi frkagemlgr hivavlpeid
    301 pvlfqgkigl pilcvgsvwk swellkegfl laltqgreiq aqnffssftl mklrhssalg
    361 gaslgarhig hllpmdysan aiafysytfs
    N-acetylglucosamine kinase, isoform 2, NP_001317354.1, NP_001317355.1 (SEQ ID
    NO: 117)
    1 mvnrakrkag vdplvplrsl glslsggdqe dagrilieel rdrfpylses ylittdaags
    61 iatatpdggv vlisgtgsnc rlinpdgses gcggwghmmg degsaywiah qavkivfdsi
    121 dnleaaphdi gyvkqamfhy fqvpdrlgil thlyrdfdkc rfagfcrkia egaqqgdpls
    181 ryifrkagem lgrhivavlp eidpvlfqgk iglpilcvgs vwkswellke gfllaltqgr
    241 eiqaqnffss ftlmklrhss alggaslgar highllpmdy sanaiafysy tfs
    Napsin A aspartic peptidase, preproprotein, NP_004842.1 (SEQ ID NO: 118)
    1 mspppllqpl llllpllnve psgatlirip lhrvqpgrri lnllrgwrep aelpklgaps
    61 pgdkpifvpl snyrdvqyfg eiglgtppqn ftvafdtgss nlwvpsrrch ffsvpcwlhh
    121 rfdpkasssf qangtkfaiq ygtgrvdgil sedkltiggi kgasvifgea lwepslvfaf
    181 ahfdgilglg fpilsvegvr ppmdvlveqg lldkpvfsfy lnrdpeepdg gelvlggsdp
    241 ahyippltfv pvtvpaywqi hmervkvgpg ltlcakgcaa ildtgtslit gpteeiralh
    301 aaiggiplla geyiilcsei pklpavsfll ggvwfnltah dyviqttrng vrlclsgfqa
    361 ldvpppagpf wilgdvflgt yvavfdrgdm kssarvglar artrgadlgw getaqaqfpg
    Nuclear transcription factor Y subunit gamma, isoform 1, NP_001136060.1 (SEQ
    ID NO: 119)
    1 msteggfggt sssdaqqslq sfwprvmeei rnltvkdfrv qelplarikk imkldedvkm
    61 isaeapvlfa kaaqifitel tlrawihted nkrrtlqrnd iamaitkfdq fdflidivpr
    121 delkppkrqe evrqsvtpae pvqyyftlaq qptavqvqgq qqgqqttsst ttiqpgqiii
    181 aqpqqgqttp vtmqvgegqq vqivqaqpqg qaqqaqsgtg qtmqvmqqii tntgeiqqip
    241 vqlnagqlqy irlaqpvsgt qvvqgqiqtl atnaqqgqrn asqgkprrcl ketlqitqte
    301 vqqgqqqfsq ftdgqqlyqi qqvtmpagqd laqpmfiqsa nqpsdgqapq vtgd
    Nuclear transcription factor Y subunit gamma, isoform 2, NP_055038.2 (SEQ ID
    NO: 120)
    1 msteggfggt sssdaqqslq sfwprvmeei rnltvkdfrv qelplarikk imkldedvkm
    61 isaeapvlfa kaaqifitel tlrawihted nkrrtlqrnd iamaitkfdq fdflidivpr
    121 delkppkrqe evrqsvtpae pvqyyftlaq qptavqvqgq qqgqqttsst ttiqpgqiii
    181 aqpqqgqttp vtmqvgegqq vqivqaqpqg qaqqaqsgtg qtmqvmqqii tntgeiqqip
    241 vqlnagqlqy irlaqpvsgt qvvqgqiqtl atnaqqitqt evqqgqqqfs qftdgqqlyq
    301 iqqvtmpagq dlaqpmfigs anqpsdgqap qvtgd
    Nuclear transcription factor Y subunit gamma, isoform 3, NP_001136059.1 (SEQ
    ID NO: 121)
    1 msteggfggt sssdaqqslq sfwprvmeei rnltvkdfrv qelplarikk imkldedvkm
    61 isaeapvlfa kaaqifitel tlrawihted nkrrtlqrnd iamaitkfdq fdflidivpr
    121 delkppkrqe evrqsvtpae pvqyyftlaq qptavqvqgq qqgqqttsst ttiqpgqiii
    181 aqpqqgqttp vtmqvgegqq vqivqaqpqg qaqqaqsgtg qtmqvmqqii tntgeiggip
    241 vqlnagqlqy irlaqpvsgt qvvqgqiqtl atnaqqitqt evqqgqqqfs qftdgqlyqi
    301 qqvtmpagqd laqpmfiqsa nqpsdgqapq vtgd
    Nuclear transcription factor Y subunit gamma, isoform 4, NP_001136061.1 (SEQ
    ID NO: 122)
    1 msteggfggt sssdaqqslq sfwprvmeei rnltvkdfrv qelplarikk imkldedvkr
    61 ndiamaitkf dqfdflidiv prdelkppkr qeevrqsvtp aepvqyyftl aqqptavqvq
    121 gqqqgqqtts stttiqpgqi iiaqpqqgqt tpvtmqvgeg qqvqivqaqp qgqaqqaqsg
    181 tgqtmqvmqq iitntgeiqq ipvqlnagql gyirlaqpvs gtqvvqgqiq tlatnaqqit
    241 qtevqqgqqq fsqftdgqql yqiqqvtmpa gqdlaqpmfi qsanqpsdgq apqvtgd
    Nuclear transcription factor Y subunit gamma, isoform 5, NP_001136062.1 (SEQ
    ID NO: 123)
    1 msteggfggt sssdaqqslq sfwprvmeei rnltvkdfrv qelplarikk imkldedvkm
    61 isaeapvlfa kaaqifitel tlrawihted nkrrtlqrnd iamaitkfdq fdflidivpr
    121 delkppkrqe evrqsvtpae pvqyyftlaq qptavqvqgq qqgqqttsst ttiqpgqiii
    181 aqpqqgqtmq vmqqiitntg eiqqipvqln agqlqyirla qpvsgtqvvg gqiqtlatna
    241 qqitqtevqq gqqqfsqftd gqqlyqiqqv tmpagqdlaq pmfiqsanqp sdgqapqvtg
    301 d
    Nuclear transcription factor Y subunit gamma, isoform 6, NP_001295043.1 (SEQ
    ID NO: 124)
    1 msteggfggt sssdaqqslq sfwprvmeei rnltvkdfrv qelplarikk imkldedvkm
    61 isaeapvlfa kaaqifitel tlrawihted nkrrtlqrnd iamaitkfdq fdflidivpr
    121 delkppkrqe evrqsvtpae pvqyyftlaq qptavqvqgq qqgqqttsst ttiqpgqiii
    181 aqpqqgqttp vtmqvgegqq vqivqaqpqg qaqqaqsgtg qtmqvmqqii tntgeiqqip
    241 vqlnagqlqy irlaqpvsgt qvvqgqiqtl atnaqqgqrn asqgkprrcl ketlqitqte
    301 vqqgqqqfsq ftdgqrnsvq qarvseltge aeprevkatg nstpctsslp tthppshrag
    361 ascvccsqpq qsstspppsd alqwvvvevs gtpnqlethr elhaplpgmt slsplhpsqq
    421 lyqiqqvtmp agqdlaqpmf iqsanqpsdg qapqvtgd
    Nuclear transcription factor Y subunit gamma, isoform 7, NP_001295044.1 (SEQ
    ID NO: 125)
    1 msteggfggt sssdaqqslq sfwprvmeei rnltvkdfrv qelplarikk imkldedvkm
    61 isaeapvlfa kaaqifitel tlrawihted nkrrtlqrnd iamaitkfdq fdflidivpr
    121 delkppkrqe evrqsvtpae pvqyyftlaq qptavqvqgq qqgqqttsst ttiqpgqiii
    181 aqpqqgqttp vtmqvgegqq vqivqaqpqg qaqqaqsgtg qtmqvmqqii tntgeiqqip
    241 vqlnagqlqy irlaqpvsgt qvvqgqiqtl atnaqqitqt evqqgqqqfs qftdgqrnsv
    301 qqarvseltg eaeprevkat gnstpctssl ptthppshra gascvccsqp qqsstsppps
    361 dalqwvvvev sgtpngleth relhaplpgm tslsplhpsq qlyqiqqvtm pagqdlaqpm
    421 fiqsanqpsd gqapqvtgd
    NFKB repressing factor, isoform 1, NP_001166958.1 (SEQ ID NO: 126)
    1 mgfmlplifr ysprlmekil qmaegidige mpsydlvlsk pskgqkrhls tcdgqnppkk
    61 qagskfharp rfepvhfvas sskderqedp ygpqtkevne qthfasmprd iygdytqdsf
    121 siqdgnsqyc dssgfiltkd qpvtanmyfd sgnpapstts qqansqstpe pspsqtfpes
    181 vvaekqyfie kltatiwknl snpemtsgsd kinytymltr ciqacktnpe yiyaplkeip
    241 padipknkkl ltdgyacevr cqniylttgy agskngsrdr atelavkllq krievrvvrr
    301 kfkhtfgedl vvcqigmssy efppalkppe dlvvlgkdas gqpifnasak hwtnfviten
    361 andaigilnn sasfnkmsie ykyemmpnrt wrcrvflqdh claegygtkk tskhaaadea
    421 lkilqktqpt ypsvkssqch tgssprgsgk kkdikdlvvy enssnpvctl ndtaqfnrmt
    481 veyvyermtg lrwkckvile seviaeavgv kktvkyeaag eavktlkktq ptvinnlkkg
    541 avedvisrne iqgrsaeeay kqqikednig nqllrkmgwt ggglgksgeg irepisvkeq
    601 hkreglgldv ervnkiakrd ieqiirnyar seshtdltfs reltnderkq ihqiaqkygl
    661 kskshgvghd rylvvgrkrr kedlldqlkq egqvghyelv mpqan
    NFKB repressing factor, isoform 2, NP_001166959.1, NP_060014.2 (SEQ ID NO:
    127)
    1 mekilqmaeg idigempsyd lvlskpskgq krhlstcdgq nppkkqagsk fharprfepv
    61 hfvassskde rqedpygpqt kevneqthfa smprdiyqdy tqdsfsiqdg nsqycdssgf
    121 iltkdqpvta nmyfdsgnpa psttsqqans qstpepspsq tfpesvvaek qyfiekltat
    181 iwknlsnpem tsgsdkinyt ymltrciqac ktnpeyiyap lkeippadip knkklltdgy
    241 acevrcqniy lttgyagskn gsrdratela vkllqkriev rvvrrkfkht fgedlvvcqi
    301 gmssyefppa lkppedlvvl gkdasgqpif nasakhwtnf vitenandai gilnnsasfn
    361 kmsieykyem mpnrtwrcrv flqdhclaeg ygtkktskha aadealkilq ktqptypsvk
    421 ssqchtgssp rgsgkkkdik dlvvyenssn pvctlndtaq fnrmtveyvy ermtglrwkc
    481 kvilesevia eavgvkktvk yeaageavkt lkktqptvin nlkkgavedv isrneiqgrs
    541 aeeaykqqik ednignqllr kmgwtggglg ksgegirepi svkeqhkreg lgldvervnk
    601 iakrdieqii rnyarsesht dltfsreltn derkqihqia qkyglksksh gvghdrylvv
    661 grkrrkedll dqlkqegqvg hyelvmpqan
    Plasminogen activator, urokinase, urokinase-type plasminogen activator
    isoform 1 preproprotein, NP_002649.1 (SEQ ID NO: 128)
    1 mrallarlll cvlvvsdskg snelhqvpsn cdclnggtcv snkyfsnihw cncpkkfggq
    61 hceidksktc yegnghfyrg kastdtmgrp clpwnsatvl qqtyhahrsd alqlglgkhn
    121 ycrnpdnrrr pwcyvqvglk plvqecmvhd cadgkkpssp peelkfqcgq ktlrprfkii
    181 ggefttienq pwfaaiyrrh rggsvtyvcg gslispcwvi sathcfidyp kkedyivylg
    241 rsrinsntqg emkfevenli lhkdysadtl ahhndiallk irskegrcaq psrtiqticl
    301 psmyndpqfg tsceitgfgk enstdylype qlkmtvvkli shrecqqphy ygsevttkml
    361 caadpqwktd scqgdsggpl vcslqgrmtl tgivswgrgc alkdkpgvyt rvshflpwir
    421 shtkeengla l
    Plasminogen activator, urokinase, urokinase-type plasminogen activator
    isoform 2, NP_001138503.1 (SEQ ID NO: 129)
    1 mvfhlrtrye qancdclngg tcvsnkyfsn ihwcncpkkf ggqhceidks ktcyegnghf
    61 yrgkastdtm grpclpwnsa tvlqqtyhah rsdalqlglg khnycrnpdn rrrpwcyvqv
    121 glkplvqecm vhdcadgkkp ssppeelkfq cgqktlrprf kiiggeftti enqpwfaaiy
    181 rrhrggsvty vcggslispc wvisathcfi dypkkedyiv ylgrsrlnsn tqgemkfeve
    241 nlilhkdysa dtlahhndia llkirskegr caqpsrtiqt iclpsmyndp qfgtsceitg
    301 fgkenstdyl ypeqlkmtvv klishrecqq phyygsevtt kmlcaadpqw ktdscqgdsg
    361 gplvcslqgr mtltgivswg rgcalkdkpg vytrvshflp wirshtkeen glal
    Plasminogen activator, urokinase, urokinase-type plasminogen activator
    isoform 3, NP_001306120.1 (SEQ ID NO: 130)
    1 mgrpclpwns atvlqqtyha hrsdalqlgl gkhnycrnpd nrrrpwcyvq vglkplvqec
    61 mvhdcadgkk pssppeelkf qcgqktlrpr fkiiggeftt ienqpwfaai yrrhrggsvt
    121 yvcggslisp cwvisathcf idypkkedyi vylgrsrlns ntqgemkfev enlilhkdys
    181 adtlahhndi allkirskeg rcaqpsrtiq ticlpsmynd pqfgtsceit gfgkenstdy
    241 lypeqlkmtv vklishrecq qphyygsevt tkmlcaadpq wktdscqgds ggplvcslqg
    301 rmtltgivsw grgcalkdkp gvytrvshfl pwirshtkee nglal
    Receptor tyrosine kinase like orphan receptor 1, inactive tyrosine-protein
    kinase transmembrane receptor ROR1 isoform 1 precursor, NP_005003.2 (SEQ ID
    NO: 131)
    1 mhrprrrgtr ppllallaal llaargaaaq etelsysael vptsswniss elnkdsyltl
    61 depmnnitts lgqtaelhck vsgnppptir wfkndapvvq eprrlsfrst iygsrlrirn
    121 ldttdtgyfq cvatngkevv sstgvlfvkf gppptaspgy sdeyeedgfc qpyrgiacar
    181 fignrtvyme slhmqgeien qitaaftmig tsshlsdkcs qfaipslchy afpycdetss
    241 vpkprdlcrd eceilenvlc qteyifarsn pmilmrlklp ncedlpqpes peaancirig
    301 ipmadpinkn hkcynstgvd yrgtvsvtks grqcqpwnsq yphthtftal rfpelngghs
    361 ycrnpgnqke apwcftlden fksdlcdipa cdskdskekn kmeilyilvp svaiplaial
    421 lffficvcrn nqksssapvq rqpkhvrgqn vemsmlnayk pkskakelpl savrfmeelg
    481 ecafgkiykg hlylpgmdha qlvaiktlkd ynnpqqwtef qqeaslmael hhpnivcllg
    541 avtqeqpvcm lfeyinqgdl heflimrsph sdvgcssded gtvkssldhg dflhiaiqia
    601 agmeylsshf fvhkdlaarn iligeqlhvk isdlglsrei ysadyyrvqs ksllpirwmp
    661 peaimygkfs sdsdiwsfgv vlweifsfgl qpyygfsnqe viemvrkrql lpcsedcppr
    721 myslmtecwn eipsrrprfk dihvrlrswe glsshtsstt psggnattqt tslsaspvsn
    781 lsnprypnym fpsqgitpqg qiagfigppi pqnqrfipin gypippgyaa fpaahyqptg
    841 pprviqhcpp pksrspssas gststghvts lpssgsnqea nipllphmsi pnhpggmgit
    901 vfgnksqkpy kidskqasll gdanihghte smisael
    Receptor tyrosine kinase like orphan receptor 1, inactive tyrosine-protein
    kinase transmembrane receptor ROR1 isoform 2 precursor, NP_001077061.1 (SEQ
    ID NO: 132)
    1 mhrprrrgtr ppllallaal llaargaaaq etelsvsael vptsswniss elnkdsyltl
    61 depmnnitts lgqtaelhck vsgnppptir wfkndapvvq eprrlsfrst iygsrlrirn
    121 ldttdtgyfq cvatngkevv sstgvlfvkf gppptaspgy sdeyeedgfc qpyrgiacar
    181 fignrtvyme slhmqgeien qitaaftmig tsshlsdkcs qfaipslchy afpycdetss
    241 vpkprdlcrd eceilenvlc qteyifarsn pmilmrlklp ncedlpqpes peaancirig
    301 ipmadpinkn hkcynstgvd yrgtvsvtks grqcqpwnsq yphthtftal rfpelngghs
    361 ycrnpgnqke apwcftlden fksdlcdipa cgk
    Runt related transcription factor 1, runt-related transcription factor 1
    isoform AML1a, NP_00lll6079.1 (SEQ ID NO: 133)
    1 mripvdasts rrftppstal spgkmsealp lgapdagaal agklrsgdrs mvevladhpg
    61 elvrtdspnf lcsvlpthwr cnktlpiafk vvalgdvpdg tlvtvmagnd enysaelrna
    121 taamknqvar fndlrfvgrs grgksftlti tvftnppqva tyhraikitv dgpreprrhr
    181 qklddqtkpg slsfserlse leqlrrtamr vsphhpaptp npraslnhst afnpqpqsqm
    241 qeedtapwrc
    Runt related transcription factor 1, runt-related transcription factor 1
    isoform AML1b, NP_001001890.1 (SEQ ID NO: 134)
    1 mripvdasts rrftppstal spgkmsealp lgapdagaal agklrsgdrs mvevladhpg
    61 elvrtdspnf lcsvlpthwr cnktlpiafk vvalgdvpdg tlvtvmagnd enysaelrna
    121 taamknqvar fndlrfvgrs grgksftlti tvftnppqva tyhraikitv dgpreprrhr
    181 qklddqtkpg slsfserlse leqlrrtamr vsphhpaptp npraslnhst afnpqpqsqm
    241 qdtrqiqpsp pwsydqsyqy lgsiaspsvh patpispgra sgmttlsael ssrlstapdl
    301 tafsdprqfp alpsisdprm hypgaftysp tpvtsgigig msamgsatry htylpppypg
    361 ssqaqggpfq asspsyhlyy gasagsyqfs mvggersppr ilppctnast gsallnpslp
    421 nqsdvveaeg shsnsptnma psarleeavw rpy
    Runt related transcription factor 1, runt-related transcription factor 1
    isoform AML1c, NP_001745.2 (SEQ ID NO: 135)
    1 masdsifesf psypqcfmre cilgmnpsrd vhdastsrrf tppstalspg kmsealplga
    61 pdagaalagk lrsgdrsmve vladhpgelv rtdspnflcs vlpthwrcnk tlpiafkvva
    121 lgdvpdgtlv tvmagndeny saelrnataa mknqvarfnd lrfvgrsgrg ksftltitvf
    181 tnppqvatyh raikitvdgp reprrhrqkl ddqtkpgsls fserlseleq lrrtamrvsp
    241 hhpaptpnpr aslnhstafn pqpqsqmqdt rqiqpsppws ydqsyqylgs iaspsvhpat
    301 pispgrasgm ttlsaelssr lstapdltaf sdprqfpalp sisdprmhyp gaftysptpv
    361 tsgigigmsa mgsatryhty lpppypgssq aqggpfqass psyhlyygas agsyqfsmvg
    421 gerspprilp pctnastgsa llnpslpnqs dvveaegshs nsptnmapsa rleeavwrpy
    Surfactant protein A1, pulmonary surfactant-associated protein A1 isoform 1
    precursor, NP_001158116.1, NP_001158119.1, NP_005402.3 (SEQ ID NO: 136)
    1 mwlcplalnl ilmaasgavc evkdvcvgsp gipgtpgshg lpgrdgrdgl kgdpgppgpm
    61 gppgempcpp gndglpgapg ipgecgekge pgergppglp ahldeelqat lhdfrhqilq
    121 trgalslqgs imtvgekvfs sngqsitfda iqeacaragg riavprnpee neaiasfvkk
    181 yntyayvglt egpspgdfry sdgtpvnytn wyrgepagrg keqcvemytd gqwndrncly
    241 srlticef
    Surfactant protein A1, pulmonary surfactant-associated protein A1 isoform 2
    precursor, NP_001087239.2 (SEQ ID NO: 137)
    1 mrpcqvpgaa tgpramwlcp lalnlilmaa sgavcevkdv cvgspgipgt pgshglpgrd
    61 grdglkgdpg ppgpmgppge mpcppgndgl pgapgipgec gekgepgerg ppglpahlde
    121 elqatlhdfr hqilqtrgal slqgsimtvg ekvfssngqs itfdaiqeac araggriavp
    181 rnpeeneaia sfvkkyntya yvgltegpsp gdfrysdgtp vnytnwyrge pagrgkeqcv
    241 emytdgqwnd rnclysrlti cef
    Surfactant protein A1, pulmonary surfactant-associated protein A1 isoform 3
    precursor, NP_001158117.1 (SEQ ID NO: 138)
    1 mrpcqvpgaa tgpramwlcp lalnlilmaa sgavcevkdv cvgtpgipge cgekgepger
    61 gppglpahld eelqatlhdf rhqilqtrga lslqgsimtv gekvfssngq sitfdaiqea
    121 caraggriav prnpeeneai asfvkkynty ayvgltegps pgdfrysdgt pvnytnwyrg
    181 epagrgkeqc vemytdgqwn drnclysrlt icef
    Surfactant protein A1, pulmonary surfactant-associated protein A1 isoform 4
    precursor, NP_001158118.1 (SEQ ID NO: 139)
    1 mwlcplalnl ilmaasgavc evkdvcvgtp gipgecgekg epgergppgl pahldeelqa
    61 tlhdfrhqil qtrgalslqg simtvgekvf ssngqsitfd aiqeacarag griavprnpe
    121 eneaiasfvk kyntyayvgl tegpspgdfr ysdgtpvnyt nwyrgepagr gkeqcvemyt
    181 dgqwndrncl ysrlticef
    Surfactant protein A2, pulmonary surfactant-associated protein A2 isoform 1
    precursor, NP_001092138.1, NP_001307742.1 (SEQ ID NO: 140)
    1 mwlcplaltl ilmaasgaac evkdvcvgsp gipgtpgshg lpgrdgrdgv kgdpgppgpm
    61 gppgetpcpp gnnglpgapg vpgergekge agergppglp ahldeelqat lhdfrhqilq
    121 trgalslqgs imtvgekvfs sngqsitfda iqeacaragg riavprnpee neaiasfvkk
    181 yntyayvglt egpspgdfry sdgtpvnytn wyrgepagrg keqcvemytd gqwndrncly
    241 srlticef
    Surfactant protein A2, pulmonary surfactant-associated protein A2 isoform 2
    precursor, NP_001307743.1 (SEQ ID NO: 141)
    1 mpgaatgpra mwlcplaltl ilmaasgaac evkdvcvgsp gipgtpgshg lpgrdgrdgv
    61 kgdpgppgpm gppgetpcpp gnnglpgapg vpgergekge agergppglp ahldeelqat
    121 lhdfrhqilq trgalslqgs imtvgekvfs sngqsitfda iqeacaragg riavprnpee
    181 neaiasfvkk yntyayvglt egpspgdfry sdgtpvnytn wyrgepagrg keqcvemytd
    241 gqwndrncly srlticef
    Surfactant protein B, pulmonary surfactant-associated protein B precursor,
    NP_000533.3, NP_942140.2 (SEQ ID NO: 142)
    1 mhqagypgcr gamaeshllq wlllllptlc gpgtaawtts slacaqgpef wcgsleqalq
    61 cralghclqe vwghvgaddl cqecedivhi lnkmakeaif qdtmrkfleq ecnvlplkll
    121 mpqcnqvldd yfplvidyfq nqtdsngicm hlglcksrqp epeqepgmsd plpkplrdpl
    181 pdplldklvl pvlpgalqar pgphtqdlse qqfpiplpyc wlcralikri qamipkgala
    241 vavaqvcrvv plvaggicqc laerysvill dtllgrmlpq lvcrlvlrcs mddsagprsp
    301 tgewlprdse chlcmsvttq agnsseqaip qamlqacvgs wldrekckqf veghtpqllt
    361 lvprgwdaht tcgalgvcgt mssplqcihs pdl
    Surfactant protein C, pulmonary surfactant-associated protein C isoform 1
    precursor, NP_001165881.1, NP_003009.2 (SEQ ID NO: 143)
    1 mdvgskevlm esppdysaap rgrfgipccp vhlkrllivv vvvvlivvvi vgallmglhm
    61 sqkhtemvle msigapeagq rlalsehlvt tatfsigstg lvvydyqqll iaykpapgtc
    121 cyimkiapes ipslealtrk vhnfqmecsl qakpavptsk lgqaegrdag sapsggdpaf
    181 lgmaysticg evplyyi
    Surfactant protein C, pulmonary surfactant-associated protein C isoform 2
    precursor, NP_001165828.1, NP_001304707.1, NP_001304709.1 (SEQ ID NO: 144)
    1 mdvgskevlm esppdysaap rgrfgipccp vhlkrllivv vvvvlivvvi vgallmglhm
    61 sqkhtemvle msigapeaqq rlalsehlvt tatfsigstg lvvydyqqll iaykpapgtc
    121 cyimkiapes ipslealtrk vhnfqakpav ptsklgqaeg rdagsapsgg dpaflgmavs
    181 ticgevplyy i
    Surfactant protein C, pulmonary surfactant-associated protein C isoform 3
    precursor, NP_001304708.1 (SEQ ID NO: 145)
    1 mdvgskevlm esppvlemsi gapeaqqrla lsehlvttat fsigstglvv ydyqqlliay
    61 kpapgtccyi mkiapesips lealtrkvhn fqmecslqak pavptsklgq aegrdagsap
    121 sggdpaflgm aysticgevp lyyi
    Surfactant protein D, pulmonary surfactant-associated protein D precursor,
    NP_003010.4 (SEQ ID NO: 146)
    1 mllfllsalv lltqplgyle aemktyshrt mpsactlvmc ssvesglpgr dgrdgregpr
    61 gekgdpglpg aagqagmpgq agpvgpkgdn gsvgepgpkg dtgpsgppgp pgvpgpagre
    121 gplgkqgnig pqgkpgpkge agpkgevgap gmqgsagarg lagpkgergv pgergvpgnt
    181 gaagsagamg pqgspgargp pglkgdkgip gdkgakgesg lpdvaslrqg vealqgqvqh
    241 lqaafsqykk velfpngqsv gekifktagf vkpfteaqll ctqaggqlas prsaaenaal
    301 qqlvvaknea aflsmtdskt egkftyptge slvysnwapg epnddggsed cveiftngkw
    361 ndracgekrl vvcef
    Solute carrier family 2 member 5, solute carrier family 2, facilitated
    glucose transporter member 5 isoform 1, NP_001315548.1, NP_003030.1 (SEQ ID
    NO: 147)
    1 meqqdqsmke grltlvlala tliaafgssf qygynvaavn spallmqqfy netyygrtge
    61 fmedfpltll wsvtvsmfpf ggfigsllvg plvnkfgrkg allfnnifsi vpailmgcsr
    121 vatsfeliii srllvgicag vssnvvpmyl gelapknlrg algvvpqlfi tvgilvaqif
    181 glrnllanvd gwpillgltg vpaalqllll pffpespryl liqkkdeaaa kkalqtlrgw
    241 dsvdrevaei rqedeaekaa gfisvlklfr mrslrwqlls iivlmggqql sgvnaiyyya
    301 dqiylsagvp eehvqyvtag tgavnvvmtf cavfvvellg rrlllllgfs icliaccvlt
    361 aalalqdtvs wmpyisivcv isyvighalg pspipallit eiflqssrps afmvggsvhw
    421 lsnftvglif pfigeglgpy sfivfavicl lttiyifliv petkaktfie inqiftkmnk
    481 vsevypekee lkelppvtse q
    Solute carrier family 2 member 5, solute carrier family 2, facilitated
    glucose transporter member 5 isoform 2, NP_001129057.1 (SEQ ID NO: 148)
    1 meqqdqsmke grltlvlala tliaafgssf qygynvaavn spallmqqfy netyygrtge
    61 fmedfpltll wsvtvsmfpf ggfigsllvg plvnkfgrkg allfnnifsi vpailmgcsr
    121 vatsfeliii srllvgicag vssnvvpmyl gelapknlrg algvvpqlfi tvgilvaqif
    181 glrnllanvd gefrtsrehp hpftttlgpl lvfqshhhrt glsadwsllt gwmslggpsc
    241 pept
    Solute carrier family 2 member 5, solute carrier family 2, facilitated
    glucose transporter member 5 isoform 3, NP_001315549.1 (SEQ ID NO: 149)
    1 mgttwllstp qhwtgefmed fpltllwsvt vsmfpfggfi gsllvgplvn kfgrkgallf
    61 nnifsivpai lmgcsrvats feliiisrll vgicagvssn vvpmylgela pknlrgalgv
    121 vpqlfitvgi lvaqifglrn llanvdgwpi llgltgvpaa lqllllpffp esprylliqk
    181 kdeaaakkal qtlrgwdsvd revaeirqed eaekaagfis vlklfrmrsl rwqllsiivl
    241 mggqqlsgvn aiyyyadqiy lsagvpeehv qyvtagtgav nvvmtfcavf vvellgrrll
    301 lllgfsicli accvltaala lqdtvswmpy isivcvisyv ighalgpspi palliteifl
    361 qssrpsafmv ggsvhwlsnf tvglifpfiq eglgpysfiv faviclltti yiflivpetk
    421 aktfieinqi ftkmnkvsev ypekeelkel ppvtseq
    Solute carrier family 2 member 5, solute carrier family 2, facilitated
    glucose transporter member 5 isoform 4, NP_001315550.1 (SEQ ID NO: 150)
    1 mylgelapkn lrgalgvvpq lfitvgilva qifglrnlla nvdgwpillg ltgvpaalql
    61 lllpffpesp rylliqkkde aaakkalqtl rgwdsvdrev aeirqedeae kaagfisvlk
    121 lfrmrslrwq llsiivlmgg qqlsgvnaiy yyadqiylsa gvpeehvgyv tagtgavnvv
    181 mtfcavfvve llgrrlllll gfsicliacc vltaalalqd tvswmpyisi vcvisyvigh
    241 algpspipal liteiflqss rpsafmvggs vhwlsnftvg lifpfiqegl gpysfivfav
    301 icllttiyif livpetkakt fieinqiftk mnkvsevype keelkelppv tseq
    Sperm associated antigen 9, C-Jun-amino-terminal kinase-interacting protein 4
    isoform 1, NP_001124000.1 (SEQ ID NO: 151)
    1 meledgvvyq eepggsgavm servsglags iyreferlig rydeevvkel mplvvavlen
    61 ldsvfaqdqe hqvelellrd dneqlitqye rekalrkhae ekfiefedsq eqekkdlqtr
    121 veslesqtrq lelkaknyad qisrleerea elkkeynalh qrhtemihny mehlertklh
    181 qlsgsdqles tahsrirker pislgifplp agdglltpda qkggetpgse qwkfgelsqp
    241 rshtslkvsn spepqkaveq edelsdvsqg gskattpast ansdvatipt dtplkeeneg
    301 fvkvtdapnk seiskhievq vaqetrnvst gsaeneekse vqaiiestpe ldmdkdlsgy
    361 kgsstptkgi enkafdrnte slfeelssag sgligdvdeg adllgmgrev enlilentql
    421 letknalniv kndliakvde ltcekdvlqg eleavkqakl kleeknrele eelrkaraea
    481 edarqkakdd ddsdiptaqr krftrvemar vlmernqyke rlmelqeavr wtemirasre
    541 npamgekkrs siwqffsrlf ssssnttkkp eppvnlkyna ptshvtpsvk krsstlsqlp
    601 gdkskafdfl seeteaslas rreqkreqyr qvkahvqked grvqafgwsl pqkykqvtng
    661 qgenkmknlp vpvylrplde kdtsmklwca vgvnlsggkt rdggsvvgas vfykdvagld
    721 tegskqrsas gssldkldge lkeqqkelkn qeelsslvwi ctsthsatkv liidavqpgn
    781 ildsftvcns hvlciasvpg aretdypage dlsesgqvdk aslcgsmtsn ssaetdsllg
    841 gitvvgcsae gvtgaatsps tngaspvmdk ppemeaense vdenvptaee ateategnag
    901 saedtvdisq tgvytehvft dplgvqiped lspvyqssnd sdaykdqisv lpneqdlvre
    961 eaqkmssllp tmwlgaqngc lyvhssvaqw rkclhsiklk dsilsivhvk givlvaladg
    1021 tlaifhrgvd gqwdlsnyhl ldlgrphhsi rcmtvvhdkv wcgyrnkiyv vqpkamkiek
    1081 sfdahprkes qvrqlawvgd gvwvsirlds tlrlyhahty qhlqdvdiep yvskmlgtgk
    1141 lgfsfvrita lmvscnrlwv gtgngviisi pltetnktsg vpgnrpgsvi rvygdensdk
    1201 vtpgtfipyc smahaqlcfh ghrdavkffv avpgqvispq ssssgtdltg dkagpsaqep
    1261 gsqtplksml visggegyid frmgdegges ellgedlple psvtkaersh livwqvmygn
    1321 e
    Sperm associated antigen 9, C-Jun-amino-terminal kinase-interacting protein 4
    isoform 2, NP_001123999.1 (SEQ ID NO: 152)
    1 meledgvvyq eepggsgavm servsglags iyreferlig rydeevvkel mplvvavlen
    61 ldsvfaqdqe hqvelellrd dneqlitqye rekalrkhae ekfiefedsq eqekkdlqtr
    121 veslesqtrq lelkaknyad qisrleerea elkkeynalh qrhtemihny mehlertklh
    181 qlsgsdqles tahsrirker pislgifplp agdglltpda qkggetpgse qwkfgelsqp
    241 rshtslkdel sdvsqggska ttpastansd vatiptdtpl keenegfvkv tdapnkseis
    301 khievqvaqe trnvstgsae neeksevqai iestpeldmd kdlsgykgss tptkgienka
    361 fdrnteslfe elssagsgli gdvdegadll gmgrevenli lentqlletk nalnivkndl
    421 iakvdeltce kdvlqgelea vkqaklklee knreleeelr karaeaedar qkakddddsd
    481 iptaqrkrft rvemarvlme rnqykerlme lqeavrwtem irasrenpam qekkrssiwq
    541 fvptrfsrlf ssssnttkkp eppvnlkyna ptshvtpsvk krsstlsqlp gdkskafdfl
    601 seeteaslas rreqkreqyr qvkahvqked grvqafgwsl pqkykqvtng qgenkmknlp
    661 vpvylrplde kdtsmklwca vgvnlsggkt rdggsvvgas vfykdvagld tegskqrsas
    721 qssldkldqe lkeqqkelkn qeelsslvwi ctsthsatkv liidavqpgn ildsftvcns
    781 hvlciasvpg aretdypage dlsesgqvdk aslcgsmtsn ssaetdsllg gitvvgcsae
    841 gvtgaatsps tngaspvmdk ppemeaense vdenvptaee ateategnag saedtvdisq
    901 tgvytehvft dplgvqiped lspvyqssnd sdaykdqisv lpneqdlvre eaqkmssllp
    961 tmwlgaqngc lyvhssvaqw rkclhsiklk dsilsivhvk givlvaladg tlaifhrgvd
    1021 gqwdlsnyhl ldlgrphhsi rcmtvvhdkv wcgyrnkiyv vqpkamkiek sfdahprkes
    1081 qvrqlawvgd gvwvsirlds tlrlyhahty qhlqdvdiep yvskmlgtgk lgfsfvrita
    1141 lmvscnrlwv gtgngviisi pltetnktsg vpgnrpgsvi rvygdensdk vtpgtfipyc
    1201 smahaqlcfh ghrdavkffv avpgqvispq ssssgtdltg dkagpsaqep gsqtplksml
    1261 visggegyid frmgdegges ellgedlple psvtkaersh livwqvmygn e
    Sperm associated antigen 9, C-Jun-amino-terminal kinase-interacting protein 4
    isoform 3, NP_003962.3 (SEQ ID NO: 153)
    1 meledgvvyq eepggsgavm servsglags iyreferlig rydeevvkel mplvvavlen
    61 ldsvfaqdqe hqvelellrd dneqlitqye rekalrkhae ekfiefedsq eqekkdlqtr
    121 veslesqtrq lelkaknyad qisrleerea elkkeynalh qrhtemihny mehlertklh
    181 qlsgsdqles tahsrirker pislgifplp agdglltpda qkggetpgse qwkfgelsqp
    241 rshtslkdel sdvsqggska ttpastansd vatiptdtpl keenegfvkv tdapnkseis
    301 khievqvaqe trnvstgsae neeksevqai iestpeldmd kdlsgykgss tptkgienka
    361 fdrnteslfe elssagsgli gdvdegadll gmgrevenli lentqlletk nalnivkndl
    421 iakvdeltce kdvlqgelea vkqaklklee knreleeelr karaeaedar qkakddddsd
    481 iptaqrkrft rvemarvlme rnqykerlme lqeavrwtem irasrenpam qekkrssiwq
    541 ffsrlfssss nttkkpeppv nlkynaptsh vtpsvkkrss tlsqlpgdks kafdflseet
    601 easlasrreq kreqyrqvka hvqkedgrvq afgwslpqky kqvtngqgen kmknlpvpvy
    661 lrpldekdts mklwcavgvn lsggktrdgg svvgasvfyk dvagldtegs kqrsasgssl
    721 dkldqelkeq qkelknqeel sslvwictst hsatkvliid avqpgnilds ftvcnshvlc
    781 iasvpgaret dypagedlse sgqvdkaslc gsmtsnssae tdsllggitv vgcsaegvtg
    841 aatspstnga spvmdkppem eaensevden vptaeeatea tegnagsaed tvdisqtgvy
    901 tehvftdplg vqipedlspv yqssndsday kdqisvlpne qdlvreeaqk mssllptmwl
    961 gaqngclyvh ssvaqwrkcl hsiklkdsil sivhvkgivl valadgtlai fhrgvdgqwd
    1021 lsnyhlldlg rphhsircmt vvhdkvwcgy rnkiyvvqpk amkieksfda hprkesqvrq
    1081 lawvgdgvwv sirldstlrl yhahtyqhlq dvdiepyvsk mlgtgklgfs fvritalmvs
    1141 cnrlwvgtgn gviisiplte tnktsgvpgn rpgsvirvyg densdkvtpg tfipycsmah
    1201 aqlcfhghrd avkffvavpg qvispqssss gtdltgdkag psaqepgsqt plksmlvisg
    1261 gegyidfrmg deggesellg edlplepsvt kaershlivw qvmygne
    Sperm associated antigen 9, C-Jun-amino-terminal kinase-interacting protein 4
    isoform 4, NP_001238900.1 (SEQ ID NO: 154)
    1 mspgcmllfv fgfvggavvi nsailvslsv lllvhfsist gvpaltqnlp rilrkerpis
    61 lgifplpagd glltpdaqkg getpgseqwk fqelsqprsh tslkdelsdv sqggskattp
    121 astansdvat iptdtplkee negfvkvtda pnkseiskhi evqvaqetrn vstgsaenee
    181 ksevqaiies tpeldmdkdl sgykgsstpt kgienkafdr nteslfeels sagsgligdv
    241 degadllgmg revenlilen tqlletknal nivkndliak vdeltcekdv lqgeleavkq
    301 aklkleeknr eleeelrkar aeaedarqka kddddsdipt aqrkrftrve marvlmernq
    361 ykerlmelqe avrwtemira srenpamqek krssiwqffs rlfssssntt kkpeppvnlk
    421 ynaptshvtp svkkrsstls qlpgdkskaf dflseeteas lasrreqkre qyrqvkahvq
    481 kedgrvqafg wslpqkykqv tngqgenkmk nlpvpvylrp ldekdtsmkl wcavgvnlsg
    541 gktrdggsvv gasvfykdva gldtegskqr sasqssldkl dqelkeqqke lknqeelssl
    601 vwictsthsa tkvliidavq pgnildsftv cnshvlcias vpgaretdyp agedlsesgq
    661 vdkaslcgsm tsnssaetds llggitvvgc saegvtgaat spstngaspv mdkppemeae
    721 nsevdenvpt aeeateateg nagsaedtvd isqtgvyteh vftdplgvqi pedlspvyqs
    781 sndsdaykdq isvlpneqdl vreeaqkmss llptmwlgaq ngclyvhssv aqwrkclhsi
    841 klkdsilsiv hvkgivlval adgtlaifhr gvdgqwdlsn yhlldlgrph hsircmtvvh
    901 dkvwcgyrnk iyvvqpkamk ieksfdahpr kesqvrqlaw vgdgvwvsir ldstlrlyha
    961 htyqhlqdvd iepyvskmlg tgklgfsfvr italmvscnr lwvgtgngvi isipltetvi
    1021 lhqgrllglr anktsgvpgn rpgsvirvyg densdkvtpg tfipycsmah aqlcfhghrd
    1081 avkffvavpg qvispqssss gtdltgdkag psaqepgsqt plksmlvisg gegyidfrmg
    1141 deggesellg edlplepsvt kaershlivw qvmygne
    SGT1 homolog, MIS12 kinetochore complex assembly cochaperone, protein SGT1
    homolog isoform A, NP_006695.1 (SEQ ID NO: 155)
    1 maaaaagtat sqrffqsfsd alidedpqaa leeltkaleq kpddaqyycq raychillgn
    61 ycvavadakk slelnpnnst amlrkgicey heknyaaale tftegqklds adanfsvwik
    121 rcqeaqngse sevwthqski kydwyqtesq vvitlmiknv qkndvnvefs ekelsalvkl
    181 psgedynlkl ellhpiipeq stfkvlstki eiklkkpeav rweklegqgd vptpkqfvad
    241 vknlypsssp ytrnwdklvg eikeeeknek legdaalnrl fqqiysdgsd evkramnksf
    301 mesggtvlst nwsdvgkrkv einppddmew kky
    SGT1 homolog, MIS512 kinetochore complex assembly cochaperone, protein SGT1
    homolog isoform B, NP_001124384.1 (SEQ ID NO: 156)
    1 maaaaagtat sqrffqsfsd alidedpqaa leeltkaleq kpddaqyycq raychillgn
    61 ycvavadakk slelnpnnst amlrkgicey heknyaaale tftegqkldi etgfhrvgqa
    121 glqlltssdp paldsqsagi tgadanfsvw ikrcqeaqng sesevwthqs kikydwyqte
    181 sqvvitlmik nvqkndvnve fsekelsalv klpsgedynl klellhpiip eqstfkvlst
    241 kieiklkkpe avrweklegq gdvptpkqfv advknlypss spytrnwdkl vgeikeeekn
    301 eklegdaaln rlfqqiysdg sdevkramnk sfmesggtvl stnwsdvgkr kveinppddm
    361 ewkky
    SGT1 homolog, MIS12 kinetochore complex assembly cochaperone, protein SGT1
    homolog isoform C, NP_001307760.1 (SEQ ID NO: 157)
    1 mlsqkevava dakkslelnp nnstamlrkg iceyheknya aaletftegq kldsadanfs
    61 vwikrcqeaq ngsesevwth qskikydwyq tesqvvitlm iknvqkndvn vefsekelsa
    121 lvklpsgedy nlklellhpi ipeqstfkvl stkieiklkk peavrwekle gqgdvptpkq
    181 fvadvknlyp ssspytrnwd klvgeikeee kneklegdaa lnrlfqqiys dgsdevkram
    241 nksfmesggt vlstnwsdvg krkveinppd dmewkky
    Sulfotransferase family 1C member 2, sulfotransferase 1C2 isoform a,
    NP_001047.1 (SEQ ID NO: 158)
    1 maltsdlgkq iklkevegtl lqpatvdnws qiqsfeakpd dllictypka gttwiqeivd
    61 mieqngdvek cqraiiqhrh pfiewarppq psgvekakam psprilkthl stqllppsfw
    121 ennckflyva rnakdcmvsy yhfqrmnhml pdpgtweeyf etfingkvvw gswfdhvkgw
    181 wemkdrhqil flfyedikrd pkheirkvmq fmgkkvdetv ldkivqetsf ekmkenpmtn
    241 rstvsksild qsissfmrkg tvgdwknhft vaqnerfdei yrrkmegtsi nfcmel
    Sulfotransferase family 1C member 2, sulfotransferase 1C2 isoform b,
    NP_789795.1 (SEQ ID NO: 159)
    1 maltsdlgkq iklkevegtl lqpatvdnws qiqsfeakpd dllictypka gttwiqeivd
    61 mieqngdvek cqraiiqhrh pfiewarppq psetgfhhva qaglkllsss nppastsqsa
    121 kitdllppsf wennckflyv arnakdcmvs yyhfqrmnhm lpdpgtweey fetfingkvv
    181 wgswfdhvkg wwemkdrhqi lflfyedikr dpkheirkvm qfmgkkvdet vldkivqets
    241 fekmkenpmt nrstvsksil dqsissfmrk gtvgdwknhf tvaqnerfde iyrrkmegts
    301 infcmel
    Transmembrane protein 52B, isoform 1, NP_694567.1 (SEQ ID NO: 160)
    1 mswrpqpcci sscclttdwv hlwyiwllvv igallllcgl tslcfrcccl srqqngedgg
    61 pppcevtvia fdhdstlqst itslqsvfgp aarrilavah shsslgqlps sldtlpgyee
    121 alhmsrftva mcgqkapdlp pvpeekqlpp tekestrivd swn
    Transmembrane protein 52B, isoform 2 precursor, NP_001073283.1 (SEQ ID NO:
    161)
    1 mgvrvhvvaa sallyfills gtrceencgn pehclttdwv hlwyiwllvv igallllcgl
    61 tslcfrcccl srqqngedgg pppcevtvia fdhdstlqst itslqsvfgp aarrilavah
    121 shsslgqlps sldtlpgyee alhmsrftva mcgqkapdlp pvpeekqlpp tekestrivd
    181 swn
    Exportin 7, NP_055839.3 (SEQ ID NO: 162)
    1 madhvqslaq lenlckqlye ttdtttrlqa ekalveftns pdclskcqll lergsssysq
    61 llaatcltkl vsrtnnplpl eqridirnyv lnylatrpkl atfvtqaliq lyaritklgw
    121 fdcqkddyvf rnaitdvtrf lqdsveycii gvtilsqltn einqadtthp ltkhrkiass
    181 frdsslfdif tlscnllkqa sgknlnlnde sqhgllmqll klthnclnfd figtstdess
    241 ddlctvqipt swrsafldss tlqlffdlyh sippsfsplv lsclvqiasv rrslfnnaer
    301 akflshlvdg vkrilenpqs lsdpnnyhef crllarlksn yqlgelvkve nypevirlia
    361 nftvtslqhw efapnsvhyl lslwqrlaas vpyvkateph mletytpevt kayitsrles
    421 vhiilrdgle dpledtglvq qqldqlstig rceyektcal lvqlfdqsaq syqellqsas
    481 aspmdiavqe grltwlvyii gaviggrvsf astdeqdamd gelvcrvlql mnltdsrlaq
    541 agneklelam lsffeqfrki yigdqvqkss klyrrlsevl glndetmvls vfigkiitnl
    601 kywgrcepit sktlqllndl sigyssvrkl vklsavqfml nnhtsehfsf lginnqsnlt
    661 dmrcrttfyt algrllmvdl gededqyeqf mlpltaafea vaqmfstnsf neqeakrtlv
    721 glvrdlrgia fafnaktsfm mlfewiypsy mpilqraiel wyhdpacttp vlklmaelvh
    781 nrsqrlqfdv sspngillfr etskmitmyg nriltlgevp kdqvyalklk gisicfsmlk
    841 aalsgsyvnf gvfrlygdda ldnalqtfik lllsiphsdl ldypklsqsy ysllevltqd
    901 hmnfiaslep hvimyilssi segltaldtm vctgccscld hivtylfkql srstkkrttp
    961 lngesdrflh imqqhpemiq qmlstvinii ifedcrnqws msrpllglil lnekyfsdlr
    1021 nsivnsqppe kqqamhlcfe nlmegiernl ltknrdrftq nlsafrrevn dsmknstygv
    1081 nsndmms
    YES proto-oncogene 1, Src family tyrosine kinase, tyrosine-protein kinase
    Yes, NP_005424.1 (SEQ ID NO: 163)
    1 mgcikskenk spaikyrpen tpepvstsvs hygaepttvs pcpsssakgt avnfsslsmt
    61 pfggssgvtp fggasssfsv vpssypaglt ggvtifvaly dyearttedl sfkkgerfqi
    121 inntegdwwe arsiatgkng yipsnyvapa dsiqaeewyf gkmgrkdaer lllnpgngrg
    181 iflvresett kgayslsird wdeirgdnvk hykirkldng gyyittraqf dtlqklvkhy
    241 tehadglchk lttvcptvkp qtqglakdaw eipreslrle vklgqgcfge vwmgtwngtt
    301 kvaiktlkpg tmmpeaflqe aqimkklrhd klvplyavvs eepiyivtef mskgslldfl
    361 kegdgkylkl pqlvdmaaqi adgmayierm nyihrdlraa nilvgenlvc kiadfglarl
    421 iedneytarq gakfpikwta peaalygrft iksdvwsfgi lqtelvtkgr vpypgmvnre
    481 vleqvergyr mpcpqgcpes lhelmnlcwk kdpderptfe yiqsfledyf tatepqyqpg
    541 enl
    Coiled-coil domain containing 80, coiled-coil domain-containing 80 precursor,
    NP_955805.1, NP_955806.1 (SEQ ID NO: 164)
    1 mtwrmgprft mllamwlvcg sephphatir gshggrkvpl vspdssrpar flrhtgrsrg
    61 ierstleepn lqplqrrrsv pvlrlarpte pparsdinga avrpeqrpaa rgspremird
    121 egssarsrml rfpsgssspn ilasfagknr vwvisaphas egyyrlmmsl lkddvycela
    181 erhiqqivlf hqageeggkv rritsegqil eqpldpslip klmsflklek gkfgmvllkk
    241 tlqveerypy pvrleamyev idqgpirrie kirqkgfvqk ckasgvegqv vaegndgggg
    301 agrpslgsek kkedprraqv pptresrvkv lrklaatapa lpqppstpra ttlppapatt
    361 vtrstsravt vaarpmttta fpttqrpwtp spshrppttt evitarrpsv senlyppsrk
    421 dqhrerpqtt rrpskatsle sftnapptti sepstraagp grfrdnrmdr rehghrdpnv
    481 vpgppkpake kppkkkaqdk ilsneyeeky dlsrptasql edelqvgnvp lkkakeskkh
    541 eklekpekek kkkmknenad kllksekqmk ksekkskqek ekskkkkggk teqdgyqkpt
    601 nkhftqspkk svadllgsfe gkrrlllita pkaennmyvq qrdeylesfc kmatrkisvi
    661 tifgpvnnst mkidhfqldn ekpmrvvdde dlvdqrlise lrkeygmtyn dffmvltdvd
    721 lrvkqyyevp itmksvfdli dtfqsrikdm ekqkkegivc kedkkqslen flsrfrwrrr
    781 llvisapnde dwaysqqlsa lsgqacnfgl rhitilkllg vgeevggvle lfpingssvv
    841 eredvpahlv kdirnyfqvs peyfsmllvg kdgnvkswyp spmwsmvivy dlidsmqlrr
    901 qemaiqqslg mrcpedeyag ygyhsyhqgy qdgyqddyrh hesyhhgypy
    Acrosin-binding protein precursor NP_115878.2 (SEQ ID NO: 165)
    1 mrkpaagflp sllkvlllpl apaaaqdstq astpgsplsp teyerffall tptwkaettc
    61 rlrathgcrn ptlvqldqye nhglvpdgav csnlpyaswf esfcqfthyr csnhvyyakr
    121 vlcsqpvsil spntlkeiea saevspttmt spisphftvt erqtfqpwpe rlsnnveell
    181 qsslslggqe qapehkqeqg vehrqeptqe hkqeegqkqe eqeeeqeeeg kqeegqgtke
    241 greaysqlqt dsepkfhses lssnpssfap rvrevestpm imeniqelir saqeidemne
    301 iydensywrn qnpgsllqlp hteallvlcy siventciit ptakawkyme eeilgfgksv
    361 cdslgrrhms tcalcdfcsl kleqchseas lqrqqcdtsh ktpfvsplla sqslsignqv
    421 gspesgrfyg ldlygglhmd fwcarlatkg cedvrvsgwl qteflsfqdg dfptkicdtd
    481 yiqypnycsf ksqqclmrnr nrkvsrmrcl qnetysalsp gksedvvlrw sqefstltlg
    541 qfg
    Alpha-fetoprotein, isoform 1 NP_001125.1 (SEQ ID NO: 166)
    1 mkwvesifli fllnftesrt lhrneygias ildsyqctae isladlatif faqfvqeaty
    61 kevskmvkda ltaiekptgd eqssgclenq lpafleelch ekeilekygh sdccsqseeg
    121 rhncflahkk ptpasiplfq vpepvtscea yeedretfmn kfiyeiarrh pflyaptill
    181 waarydkiip scckaenave cfqtkaatvt kelresslln qhacavmknf gtrtfqaitv
    241 tklsqkftkv nfteiqklvl dvahvhehcc rgdvldclqd gekimsyics qqdtlsnkit
    301 eccklttler gqciihaend ekpeglspnl nrflgdrdfn qfssgeknif lasfvheysr
    361 rhpqlavsvi lrvakgyqel lekcfqtenp lecqdkgeee lqkyiqesqa lakrscglfq
    421 klgeyylqna flvaytkkap qltsselmai trkmaataat ccqlsedkll acgegaadii
    481 ighlcirhem tpvnpgvgqc ctssyanrrp cfsslvvdet yvppafsddk fifhkdlcqa
    541 qgvalqtmkg eflinlvkqk pqiteeqlea viadfsglle kccqgqeqev cfaeegqkli
    601 sktraalgv
    Alpha-fetoprotein, isoform 2 NP_001341646.1 (SEQ ID NO: 167)
    1 mnkfiyeiar rhpflyapti llwaarydki ipscckaena vecfqtkaat vtkelressl
    61 lnqhacavmk nfgtrtfqai tvtklsqkft kvnfteiqkl vldvahvheh ccrgdvldcl
    121 qdgerimsyi csqqdtlsnk iteccklttl ergqciihae ndekpeglsp nlnrflgdrd
    181 fnqfssgekn iflasfvhey srrhpqlavs vilrvakgyq ellekcfqte nplecqdkge
    241 eelqkyiqes qalakrscgl fqklgeyylq naflvaytkk apqltsselm aitrkmaata
    301 atccqlsedk llacgegaad iiighlcirh emtpvnpgvg qcctssyanr rpcfsslvvd
    361 etyvppafsd dkfifhkdlc qaqgvalqtm kqeflinlvk qkpqiteeql eaviadfsgl
    421 lekccqgqeq evcfaeegqk lisktraalg v
    Absent in melanoma 1 protein NP_001615.2 (SEQ ID NO: 168)
    1 mplsppaqgd pgepsperpp kkhttfhlwr skkkqqpapp dcgvfvphpl papagearal
    61 dvvdgkyvvr dsqefplhcg esqffhttse algslllesg ifkksraqpp ednrrkpvlg
    121 klgtlftagr rrnsrngles ptrsnakpls pkdvvaspkl peresersrs qssqlkqtdt
    181 seegsprenp reaegelpes ggpaappdae lsprwsssaa avavqqchen dspqleplea
    241 egepfpdatt takqlhsspg nssrqenaet parspgedas pgagheqeaf lgvrgapgsp
    301 tqerpagglg eapngapsvc aeegslgprn arsqppkgas dlpgeppaeg aahtassaqa
    361 dctarpkgha hpakvltldi ylsktegaqv depvvitpra edcgdwddme krssgrrsgr
    421 rrgsqkstds pgadaelpes aarddavfdd evapnaasdn asaekkvksp raaldggvas
    481 aaspeskpsp gtkgqlrges drskqpppas sptkrkgrsr aleavpappa sgprapakes
    541 ppkrvpdpsp vtkgtaaesg eeaaraipre lpvksssllp eikpehkrgp lpnhfngrae
    601 ggrsrelgra agapgasdad glkprnhfgv grstvttkvt lpakpkhvel nlktpknlds
    661 lgnehnpfsq pvhkgntatk islfenkrtn ssprhtdirg qrntpasskt fvgraklnla
    721 kkakemeqpe kkvmpnspqn gvlvketaie tkvtvseeei lpatrgmngd ssenqalgpq
    781 pnqddkadvq tdagclsepv asalipvkdh kllekedsea adskslvlen vtdtaqdipt
    841 tvdtkdlppt ampkpqhtfs dsqspaessp gpslslsapa pgdvpkdtcv qspissfpct
    901 dlkvsenhkg cvlpvsrqnn ekmpllelgg ettpplster speavgsecp srvlvqvrsf
    961 vlpvestqdv ssqvipesse vrevqlptch snepevvsva scappqeevl gnehshctae
    1021 laaksgpqvi ppasektlpi qaqsqgsrtp lmaessptns pssgnhlatp qrpdqtvtng
    1081 qdspasllni sagsddsvfd sssdmekfte iikqmdsavc mpmkrkkarm pnspaphfam
    1141 ppihedhlek vfdpkvftfg lgkkkesqpe mspalhlmqn ldtksklrpk rasaeqsvlf
    1201 kslhtntngn seplvmpein dkenrdvtng gikrsrleks alfssllssl pqdkifspsv
    1261 tsvntmttaf stsqngslsq ssysqptteg appcglnkeq snllpdnslk vfnfnsssts
    1321 hsslkspshm ekypqkektk edldsrsnlh lpetkfsels klknddmeka nhiesviksn
    1381 lpncansdtd fmglfkssry dpsisfsgms lsdtmtlrgs vqnklnprpg kvviysepdv
    1441 sekcievfsd iqdcsswsls pvilikvvrg cwilyeqpnf eghsipleeg elelsglwgi
    1501 edilerheea esdkpvvigs irhvvqdyrv shidlftepe glgilssyfd dteemqgfgv
    1561 mqktcsmkvh wgtwliyeep gfqgvpfile pgeypdlsfw dteeayigsm rplkmggrkv
    1621 efptdpkvvv yekpffegkc veletgmcsf vmeggeteea tgddhlpfts vgsmkvlrgi
    1681 wvayekpgft ghqylleege yrdwkawggy ngelqslrpi lgdfsnahmi myseknfgsk
    1741 gssidvlgiv anlketgygv ktqsinvlsg vwvayenpdf tgeqyildkg fytsfedwgg
    1801 knckissvqp icldsftgpr rrnqihlfse pqfqghsqsf eettsqidds fstkscrvsg
    1861 gswvvydgen ftgnqyvlee ghypclsamg cppgatfksl rfidvefsep tiilferedf
    1921 kgkkielnae tvnlrslgfn tqirsvqvig giwvtyeygs yrgrqfllsp aevpnwyefs
    1981 gcrqigslrp fvqkriyfrl rnkatglfms tngnledlkl lriqvmedvg addqiwiyqe
    2041 gcikcriaed ccltivgslv tsgsklglal dqnadsqfws lksdgriysk lkpnlvldik
    2101 ggtqydqnhi ilntvskekf tqvweamvly t
    A-kinase anchoring protein 4, isoform 1 NP_003877.2 (SEQ ID NO: 169)
    1 mmaysdttmm sddidwlrsh rgvckvdlyn pegqqdqdrk vicfvdvstl nvedkdykda
    61 assssegnln lgsleekeii vikdtekkdq sktegsvclf kqapsdpvsv lnwllsdlqk
    121 yalgfqhals pststckhkv gdtegeyhra ssencysvya dqvnidylmn rpqnlrlemt
    181 aakntnnnqs psappakpps tqravispdg ecsiddlsfy vnrlsslviq mahkeikekl
    241 egkskclhhs icpspgnker isprtpaski asemayeave ltaaemrgtg eesreggqks
    301 flyselsnks ksgdkqmsqr eskefadsis kglmvyanqv asdmmvslmk tlkvhssgkp
    361 ipasvvlkrv llrhtkeivs dlidscmknl hnitgvlmtd sdfvsavkrn lfnqwkqnat
    421 dimeamlkrl vsaligeeke tksqslsyas lkagshdpkc rnqslefstm kaemkerdkg
    481 kmksdpcksl tsaekvgehi lkegltiwnq kqgnsckvat kacsnkdekg ekinastdsl
    541 akdlivsalk liqyhltqqt kgkdtceedc pgstmgymaq stqyekcggg qsakalsvkq
    601 leshrapgps tcqkenqhld sqkmdmsniv lmliqkllne npfkcedpce genkcsepra
    661 skaasmsnrs dkaeeqcqeh qeldctsgmk qangqfidkl vesvmklcli makysndgaa
    721 laeleeqaas ankpnfrgtr cihsgampqn yqdslghevi vnnqcstnsl qkqlqavlqw
    781 iaasqfnvpm lyfmgdkdgq leklpqvsak aaekgysvgg llqevmkfak erqpdeavgk
    841 varkqlldwl lanl
    A-kinase anchoring protein 4, isoform 2 NP_647450.1 (SEQ ID NO: 170)
    1 msddidwlrs hrgvckvdly npegqqdqdr kvicfvdvst lnvedkdykd aassssegnl
    61 nlgsleekei ivikdtekkd qsktegsvcl fkqapsdpvs vlnwllsdlq kyalgfqhal
    121 spststckhk vgdtegeyhr assencysvy adqvnidylm nrpqnlrlem taakntnnnq
    181 spsappakpp stqravispd gecsiddlsf yvnrlsslvi qmahkeikek legkskclhh
    241 sicpspgnke risprtpask iasemayeav eltaaemrgt geesreggqk sflyselsnk
    301 sksgdkqmsq reskefadsi skglmvyanq vasdmmvslm ktlkvhssgk pipasvvlkr
    361 vllrhtkeiv sdlidscmkn lhnitgvlmt dsdfvsavkr nlfnqwkqna tdimeamlkr
    421 lvsaligeek etksqslsya slkagshdpk crnqslefst mkaemkerdk gkmksdpcks
    481 ltsaekvgeh ilkegltiwn qkqgnsckva tkacsnkdek gekinastds lakdlivsal
    541 kliqyhltqq tkgkdtceed cpgstmgyma qstqyekcgg gqsakalsvk qleshrapgp
    601 stcqkenqhl dsqkmdmsni vlmliqklln enpfkcedpc egenkcsepr askaasmsnr
    661 sdkaeeqcqe hqeldctsgm kqangqfidk lvesvmklcl imakysndga alaeleeqaa
    721 sankpnfrgt rcihsgampq nyqdslghev ivnnqcstns lqkqlqavlq wiaasqfnvp
    781 mlyfmgdkdg qleklpqvsa kaaekgysvg gllqevmkfa kerqpdeavg kvarkqlldw
    841 llanl
    ALK tryrosine kinase receptor, isoform 1 NP_004295.2 (SEQ ID NO: 171)
    1 mgaigllwll plllstaavg sgmgtgqrag spaagpplqp replsysrlq rkslavdfvv
    61 pslfrvyard lllppsssel kagrpeargs laldcapllr llgpapgvsw tagspapaea
    121 rtlsrvlkgg svrklrrakq lvlelgeeai legcvgppge aavgllqfnl selfswwirq
    181 gegrlrirlm pekkasevgr egrlsaaira sqprllfqif gtghsslesp tnmpspspdy
    241 ftwnltwimk dsfpflshrs ryglecsfdf pceleysppl hdlrnqswsw rripseeasq
    301 mdlldgpgae rskemprgsf lllntsadsk htilspwmrs ssehctlays vhrhlqpsgr
    361 yiaqllphne aareillmpt pgkhgwtvlq grigrpdnpf rvaleyissg nrslsavdff
    421 alkncsegts pgskmalqss ftcwngtvlq lgqacdfhqd caqgedesqm crklpvgfyc
    481 nfedgfcgwt qgtlsphtpq wqvrtlkdar fqdhqdhall lsttdvpase satvtsatfp
    541 apiksspcel rmswlirgvl rgnvslvlve nktgkeqgrm vwhvaayegl slwqwmvlpl
    601 ldvsdrfwlq mvawwgqgsr aivafdnisi sldcyltisg edkilqntap ksrnlfernp
    661 nkelkpgens prqtpifdpt vhwlfttcga sgphgptqaq cnnayqnsnl svevgsegpl
    721 kgiqiwkvpa tdtysisgyg aaggkggknt mmrshgvsvl gifnlekddm lyilvgqqge
    781 dacpstnqli qkvcigennv ieeeirvnrs vhewaggggg gggatyvfkm kdgvpvplii
    841 aaggggrayg aktdtfhper lennssvlgl ngnsgaaggg ggwndntsll wagkslqega
    901 tgghscpqam kkwgwetrgg fggggggcss ggggggyigg naasnndpem dgedgvsfis
    961 plgilytpal kvmeghgevn ikhylncshc evdechmdpe shkvicfcdh gtvlaedgvs
    1021 civsptpeph lplslilsvv tsalvaalvl afsgimivyr rkhqelqamq melqspeykl
    1081 sklrtstimt dynpnycfag ktssisdlke vprknitlir glghgafgev yegqvsgmpn
    1141 dpsplqvavk tlpevcseqd eldflmeali iskfnhqniv rcigvslqsl prfillelma
    1201 ggdlksflre trprpsqpss lamldllhva rdiacgcqyl eenhfihrdi aarnclltcp
    1261 gpgrvakigd fgmardiyra syyrkggcam lpvkwmppea fmegiftskt dtwsfgvllw
    1321 eifslgympy psksnqevle fvtsggrmdp pkncpgpvyr imtqcwqhqp edrpnfaiil
    1381 erieyctqdp dvintalpie ygplveeeek vpvrpkdpeg vppllvsqqa kreeerspaa
    1441 ppplpttssg kaakkptaae isvrvprgpa vegghvnmaf sqsnppselh kvhgsrnkpt
    1501 slwnptygsw ftekptkknn piakkephdr gnlglegsct vppnvatgrl pgasllleps
    1561 sltanmkevp lfrlrhfpcg nvnygyqqqg lpleaatapg aghyedtilk sknsmnqpgp
    ALK tyrosin kinese receptor, isoform 2 NP_001340694.1 (SEQ ID NO: 172)
    1 mqmelqspey klsklrtsti mtdynpnycf agktssisdl kevprknitl irglghgafg
    61 evyegqvsgm pndpsplqva vktlpevcse qdeldflmea liiskfnhqn ivrcigvslq
    121 slprfillel maggdlksfl retrprpsqp sslamldllh vardiacgcq yleenhfihr
    181 diaarncllt cpgpgrvaki gdfgmardiy rasyyrkggc amlpvkwmpp eafmegifts
    241 ktdtwsfgvl lweifslgym pypsksnqev lefvtsggrm dppkncpgpv yrimtqcwqh
    301 qpedrpnfai ilerieyctq dpdvintalp ieygplveee ekvpvrpkdp egvppllvsq
    361 qakreeersp aappplptts sgkaakkpta aeisvrvprg pavegghvnm afsqsnppse
    421 lhkvhgsrnk ptslwnptyg swftekptkk nnpiakkeph drgnlglegs ctvppnvatg
    481 rlpgasllle pssltanmke vplfrlrhfp cgnvnygyqq qglpleaata pgaghyedti
    541 lksknsmnqp gp
    Angiopoietin-2, isoform a NP_001138.1 (SEQ ID NO: 173)
    1 mwqivfftls cdlvlaaayn nfrksmdsig kkgyqvghgs csytfllpem dncrsssspy
    61 vsnavqrdap leyddsvqrl qvlenimenn tqwlmkleny iqdnmkkemv eiqqnavqnq
    121 tavmieigtn llnqtaeqtr kltdveaqvl nqttrlelql lehslstnkl ekqildqtse
    181 inklqdknsf lekkvlamed khiiqlqsik eekdqlqvlv skqnsiieel ekkivtatvn
    241 nsvlqkqqhd lmetvnnllt mmstsnsakd ptvakeeqis frdcaevfks ghttngiytl
    301 tfpnsteeik aycdmeaggg gwtiiqrred gsvdfqrtwk eykvgfgnps geywlgnefv
    361 sqltnqqryv lkihlkdweg neayslyehf ylsseelnyr ihlkgltgta gkissisqpg
    421 ndfstkdgdn dkcickcsqm ltggwwfdac gpsnlngmyy pqrqntnkfn gikwyywkgs
    481 gyslkattmm irpadf
    Angiopoietin-2, isoform b NP_00lll2359.1 (SEQ ID NO: 174)
    1 mwqivfftls cdlvlaaayn nfrksmdsig kkqyqvghgs csytfllpem dncrsssspy
    61 vsnavqrdap leyddsvqrl qvlenimenn tqwlmkleny iqdnmkkemv eiqqnavqnq
    121 tavmieigtn llnqtaeqtr kltdveaqvl nqttrlelql lehslstnkl ekqildqtse
    181 inklqdknsf lekkvlamed khiiqlqsik eekdqlqvlv skqnsiieel ekkivtatvn
    241 nsvlqkqqhd lmetvnnllt mmstsnskdp tvakeeqisf rdcaevfksg httngiytlt
    301 fpnsteeika ycdmeagggg wtiiqrredg svdfqrtwke ykvgfgnpsg eywlgnefvs
    361 qltnqqryvl kihlkdwegn eayslyehfy lsseelnyri hlkgltgtag kissisqpgn
    421 dfstkdgdnd kcickcsqml tggwwfdacg psnlngmyyp qrqntnkfng ikwyywkgsg
    481 yslkattmmi rpadf
    Angiopoietin-2, isoform c NP_00lll2360.1 (SEQ ID NO: 175)
    1 mwqivfftls cdlvlaaayn nfrksmdsig kkgyqvqhgs csytfllpem dncrsssspy
    61 vsnavqrdap leyddsvqrl qvlenimenn tqwlmkvlnq ttrlelqlle hslstnklek
    121 qildqtsein klqdknsfle kkvlamedkh iiqlqsikee kdqlqvlvsk qnsiieelek
    181 kivtatvnns vlqkqqhdlm etvnnlltmm stsnsakdpt vakeeqisfr dcaevfksgh
    241 ttngiytltf pnsteeikay cdmeaggggw tiiqrredgs vdfqrtwkey kvgfgnpsge
    301 ywlgnefvsq ltnqqryvlk ihlkdwegne ayslyehfyl sseelnyrih lkgltgtagk
    361 issisqpgnd fstkdgdndk cickcsqmlt ggwwfdacgp snlngmyypq rqntnkfngi
    421 kwyywkgsgy slkattmmir padf
    Angiopoietin-1, isoform 1 precursor NP_001137.2 (SEQ ID NO: 176)
    1 mtvflsfafl aailthigcs nqrrspensg rrynriqhgq caytfilpeh dgncresttd
    61 gyntnalqrd aphvepdfss qklqhlehvm enytqwlqkl enyivenmks emaqiqqnav
    121 qnhtatmlei gtsllsqtae qtrkltdvet qvlnqtsrle iqllenslst yklekqllqq
    181 tneilkihek nsllehkile megkhkeeld tlkeekenlq glvtrqtyii qelekqlnra
    241 ttnnsvlqkq qlelmdtvhn lvnlctkegv llkggkreee kpfrdcadvy gagfnksgiy
    301 tiyinnmpep kkvfcnmdvn gggwtviqhr edgsldfqrg wkeykmgfgn psgeywlgne
    361 fifaitsqrq ymlrielmdw egnraysqyd rfhignekqn yrlylkghtg tagkqsslil
    421 hgadfstkda dndncmckca lmltggwwfd acgpsnlngm fytagqnhgk lngikwhyfk
    481 gpsyslrstt mmirpldf
    Angiopoietin-1, isoform 2 precursor NP_001186788.1 (SEQ ID NO: 177)
    1 mtvflsfafl aailthigcs nqrrspensg rrynriqhgq caytfilpeh dgncresttd
    61 gyntnalqrd aphvepdfss qklqhlehvm enytqwlqkl enyivenmks emaqiqqnav
    121 qnhtatmlei gtsllsqtae qtrkltdvet qvlnqtsrle iqllenslst yklekqllqq
    181 tneilkihek nsllehkile megkhkeeld tlkeekenlq glvtrqtyii qelekqlnra
    241 ttnnsvlqkq qlelmdtvhn lvnlctkevl lkggkreeek pfrdcadvyq agfnksgiyt
    301 iyinnmpepk kvfcnmdvng ggwtviqhre dgsldfqrgw keykmgfgnp sgeywlgnef
    361 ifaitsqrqy mlrielmdwe gnraysqydr fhignekqny rlylkghtgt agkqsslilh
    421 gadfstkdad ndncmckcal mltggwwfda cgpsnlngmf ytagqnhgkl ngikwhyfkg
    481 psyslrsttm mirpldf
    Angiopoietin-1, isoform 3 precursor NP_001300980.1 (SEQ ID NO: 178)
    1 megkhkeeld tlkeekenlq glvtrqtyii qelekqlnra ttnnsvlqkq qlelmdtvhn
    61 lvnlctkegv llkggkreee kpfrdcadvy qagfnksgiy tiyinnmpep kkvfcnmdvn
    121 gggwtviqhr edgsldfqrg wkeykmgfgn psgeywlgne fifaitsqrq ymlrielmdw
    181 egnraysqyd rfhignekqn yrlylkghtg tagkqsslil hgadfstkda dndncmckca
    241 lmltggwwfd acgpsnlngm fytagqnhgk lngikwhyfk gpsyslrstt mmirpldf
    Ankyrin repeat domain-containing protein 30A NP_443723.2 (SEQ ID NO: 179)
    1 mtkrkktinl niqdaqkrta lhwacvnghe evvtflvdrk cqldvldgeh rtplmkalqc
    61 hqeacanili dsgadinlvd vygntalhya vyseilsvva kllshgavie vhnkasltpl
    121 llsitkrseq ivefllikna nanavnkykc talmlavchg sseivgmllq qnvdvfaadi
    181 cgvtaehyav tcgfhhiheq imeyirklsk nhqntnpegt sagtpdeaap laertpdtae
    241 slvektpdea aplvertpdt aeslvektpd eaaslvegts dkiqclekat sgkfeqsaee
    301 tpreitspak etsekftwpa kgrprkiawe kkedtpreim spaketsekf twaakgrprk
    361 iawekketpv ktgcvarvts nktkvlekgr skmiacptke sstkasandq rfpseskqee
    421 deeyscdsrs lfessakiqv cipesiyqkv meinreveep pkkpsafkpa iemqnsvpnk
    481 afelknegtl radpmfppes kqkdyeensw dseslcetvs qkdvclpkat hqkeidking
    541 kleespnkdg llkatcgmkv siptkalelk dmqtfkaepp gkpsafepat emqksvpnka
    601 lelkneqtlr adeilpsesk qkdyeenswd teslcetvsq kdvclpkaah qkeidkingk
    661 legspvkdgl lkancgmkvs iptkalelmd mqtfkaeppe kpsafepaie mqksvpnkal
    721 elkneqtlra deilpseskq kdyeesswds eslcetvsqk dvclpkathq keidkingkl
    781 eespdndgfl kapermkvsi ptkalelmdm qtfkaeppek psafepaiem qksvpnkale
    841 lkneqtlrad qmfpseskqk kveenswdse slretvsqkd vcvpkathqk emdkisgkle
    901 dstslskild tvhscerare lqkdhceqrt gkmeqmkkkf cvlkkklsea keiksqlenq
    961 kvkweqelcs vrltlnqeee krrnadilne kireelgrie eqhrkelevk qqleqalriq
    1021 dielksvesn lnqvshthen enyllhencm lkkeiamlkl eiatlkhqyq ekenkyfedi
    1081 kilkeknael qmtlklkees ltkrasqysg qlkvliaent mltsklkekq dkeileaeie
    1141 shhprlasav qdhdqivtsr ksqepafhia gdaclqrkmn vdvsstiynn evlhqplsea
    1201 qrkskslkin lnyagdalre ntlvsehaqr dqretqcqmk eaehmyqneq dnvnkhteqq
    1261 esldqklfql qsknmwlqqq lvhahkkadn kskitidihf lerkmqhhll kekneeifny
    1321 nnhlknriyq yekekaeten s
    Androgen receptor, isoform 1 NP_000035.2 (SEQ ID NO: 180)
    1 mevqlglgrv yprppsktyr gafqnlfqsv reviqnpgpr hpeaasaapp gasllllqqq
    61 qqqqqqqqqq qqqqqqqqqq etsprqqqqq qgedgspqah rrgptgylvl deeqqpsqpq
    121 salechperg cvpepgaava askglpqqlp appdeddsaa pstlsllgpt fpglsscsad
    181 lkdilseast mqllqqqqqe aysegsssgr areasgapts skdnylggts tisdnakelc
    241 kaysysmglg vealehlspg eqlrgdcmya pllgvppavr ptpcaplaec kgsllddsag
    301 kstedtaeys pfkggytkgl egeslgcsgs aaagssgtle lpstlslyks galdeaaayq
    361 srdyynfpla lagppppppp phpharikle npldygsawa aaaaqcrygd laslhgagaa
    421 gpgsgspsaa assswhtlft aeegqlygpc gggggggggg gggggggggg gggeagavap
    481 ygytrppqgl agqesdftap dvwypggmvs rvpypsptcv ksemgpwmds ysgpygdmrl
    541 etardhvlpi dyyfppqktc licgdeasgc hygaltcgsc kvffkraaeg kqkylcasrn
    601 dctidkfrrk ncpscrlrkc yeagmtlgar klkklgnlkl qeegeasstt spteettqkl
    661 tvshiegyec qpiflnvlea iepgvvcagh dnnqpdsfaa llsslnelge rqlvhvvkwa
    721 kalpgfrnlh vddgmaviqy swmglmvfam gwrsftnvns rmlyfapdlv fneyrmhksr
    781 mysqcvrmrh lsqefgwlqi tpqeflcmka lllfsiipvd glknqkffde lrmnyikeld
    841 riiackrknp tscsrrfyql tklldsvqpi arelhqftfd llikshmvsv dfpemmaeii
    901 svqvpkilsg kvkpiyfhtq
    Androgen receptor, isoform 2 NP_001011645.1 (SEQ ID NO: 181)
    1 milwlhslet ardhvlpidy yfppqktcli cgdeasgchy galtcgsckv ffkraaegkq
    61 kylcasrndc tidkfrrknc pscrlrkcye agmtlgarkl kklgnlklqe egeassttsp
    121 teettqkltv shiegyecqp iflnvleaie pgvvcaghdn nqpdsfaall sslnelgerq
    181 lvhvvkwaka lpgfrnlhvd dqmaviqysw mglmvfamgw rsftnvnsrm lyfapdlvfn
    241 eyrmhksrmy sqcvrmrhls qefgwlqitp qeflcmkall lfsiipvdgl knqkffdelr
    301 mnyikeldri iackrknpts csrrfyqltk lldsvqpiar elhqftfdll ikshmvsvdf
    361 pemmaeiisv qvpkilsgkv kpiyfhtq
    Androgen receptor, isoform 3 NP_001334990.1 (SEQ ID NO: 182)
    1 mevqlglgrv yprppsktyr gafqnlfqsv reviqnpgpr hpeaasaapp gasllllqqq
    61 qqqqqqqqqq qqqqqqqqqq etsprqqqqq qgedgspqah rrgptgylvl deeqqpsqpq
    121 salechperg cvpepgaava askglpqqlp appdeddsaa pstlsllgpt fpglsscsad
    181 lkdilseast mqllqqqqqe aysegsssgr areasgapts skdnylggts tisdnakelc
    241 kaysysmglg vealehlspg eqlrgdcmya pllgvppavr ptpcaplaec kgsllddsag
    301 kstedtaeys pfkggytkgl egeslgcsgs aaagssgtle lpstlslyks galdeaaayq
    361 srdyynfpla lagppppppp phpharikle npldygsawa aaaaqcrygd laslhgagaa
    421 gpgsgspsaa assswhtlft aeegqlygpc gggggggggg gggggggggg gggeagavap
    481 ygytrppqgl agqesdftap dvwypggmvs rvpypsptcv ksemgpwmds ysgpygdmrl
    541 etardhvlpi dyyfppqktc licgdeasgc hygaltcgsc kvffkraaeg kqkylcasrn
    601 dctidkfrrk ncpscrlrkc yeagmtlgek frvgnckhlk mtrp
    Androgen receptor, isoform 4 NP_001334992.1 (SEQ ID NO: 183)
    1 mevqlglgrv yprppsktyr gafqnlfqsv reviqnpgpr hpeaasaapp gasllllqqq
    61 qqqqqqqqqq qqqqqqqqqq etsprqqqqq qgedgspqah rrgptgylvl deeqqpsqpq
    121 salechperg cvpepgaava askglpqqlp appdeddsaa pstlsllgpt fpglsscsad
    181 lkdilseast mqllqqqqqe aysegsssgr areasgapts skdnylggts tisdnakelc
    241 kaysvsmglg vealehlspg eqlrgdcmya pllgvppavr ptpcaplaec kgsllddsag
    301 kstedtaeys pfkggytkgl egeslgcsgs aaagssgtle lpstlslyks galdeaaayq
    361 srdyynfpla lagppppppp phpharikle npldygsawa aaaaqcrygd laslhgagaa
    421 gpgsgspsaa assswhtlft aeegqlygpc gggggggggg gggggggggg gggeagavap
    481 ygytrppqgl agqesdftap dvwypggmvs rvpypsptcv ksemgpwmds ysgpygdmrl
    541 etardhvlpi dyyfppqktc licgdeasgc hygaltcgsc kvffkraaeg kqkylcasrn
    601 dctidkfrrk ncpscrlrkc yeagmtlgaa vvvserilry fgvsewlp
    Androgen receptor, isoform 5 NP_001334993.1 (SEQ ID NO: 184)
    1 mevqlglgrv yprppsktyr gafqnlfqsv reviqnpgpr hpeaasaapp gasllllqqq
    61 qqqqqqqqqq qqqqqqqqqq etsprqqqqq qgedgspqah rrgptgylvl deeqqpsqpq
    121 salechperg cvpepgaava askglpqqlp appdeddsaa pstlsllgpt fpglsscsad
    181 lkdilseast mqllqqqqqe aysegsssgr areasgapts skdnylggts tisdnakelc
    241 kaysysmglg vealehlspg eqlrgdcmya pllgvppavr ptpcaplaec kgsllddsag
    301 kstedtaeys pfkggytkgl egeslgcsgs aaagssgtle lpstlslyks galdeaaayq
    361 srdyynfpla lagppppppp phpharikle npldygsawa aaaaqcrygd laslhgagaa
    421 gpgsgspsaa assswhtlft aeegqlygpc gggggggggg gggggggggg gggeagavap
    481 ygytrppqgl agqesdftap dvwypggmvs rvpypsptcv ksemgpwmds ysgpygdmrn
    541 trrkrlwkli irsinscics pretevpvrq qk
    ATPase H+  transporting accessory protein 1 NP_001174.2 (SEQ ID NO: 185)
    1 mmaamatarv rmgprcaqal wrmpwlpvfl slaaaaaaaa aeqqvplvlw ssdrdlwapa
    61 adtheghits dlqlstyldp alelgprnvl lflqdklsie dftayggvfg nkqdsafsnl
    121 enaldlapss lvlpavdwya vstlttylqe klgasplhvd latlrelkln aslpalllir
    181 lpytassglm aprevltgnd evigqvlstl ksedvpytaa ltavrpsrva rdvavvaggl
    241 grqllqkqpv spvihppvsy ndtaprilfw aqnfsvaykd qwedltpltf gvqelnltgs
    301 fwndsfarls ltyerlfgtt vtfkfilanr lypvsarhwf tmerlevhsn gsvayfnasq
    361 vtgpsiysfh ceyvsslskk gsllvartqp spwqmmlqdf qiqafnvmge qfsyasdcas
    421 ffspgiwmgl ltslfmlfif tyglhmilsl ktmdrfddhk gptisltqiv
    B melanoma antigen 1 precursor NP_001178.1 (SEQ ID NO: 186)
    1 maaravflal saqllqarlm keespvvswr lepedgtalc fif
    BCR/ABL fusion protein e14ab NP_050673.1 (SEQ ID NO: 187)
    1 gcacctgcag ggagggcagg cagctagcct gaaggctgat ccccccttcc tgttagcact
    61 tttgatggga ctagtggact ttggttcaga aggaagagct atgcttgtta gggcctcttg
    121 tctcctccca ggagtggaca aggtgggtta ggagcagttt ctccctgagt ggctgctgct
    181 gggtggttga ggagatgcac ggcttctgtt cctagtcaca aggctgcagc agacgctcct
    241 cagatgctct gtgccttgga tctggcccca ctcccgtcct cccagccctc ctctcctcca
    301 gctacctgcc agccggcact tttggtcaag ctgttttgca ttcactgttg cacatatgct
    361 cagtcacaca cacagcatac gctatgcaca tgtgtccaca cacaccccac ccacatccca
    421 catcaccccg accccctctg ctgtccttgg aaccttatta cacttcgagt cactggtttg
    481 cctgtattgt gaaaccagct ggatcctgag atccccaaga cagaaatcat gatgagtatg
    541 tttttggccc atgacactgg cttaccttgt gccaggcaga tggcagccac acagtgtcca
    601 ccggatggtt gattttgaag cagagttagc ttgtcacctg cctccctttc ccgggacaac
    661 agaagctgac ctctttgatc tcttgcgcag atgatgagtc tccggggctc tatgggtttc
    721 tgaatgtcat cgtccactca gccactggat ttaagcagag ttcaagtaag tactggtttg
    781 gggaggaggg ttgcagcggc cgagccaggg tctccaccca ggaaggactc atcgggcagg
    841 gtgtggggaa acagggaggt tgttcagatg accacgggac acctttgacc ctggccgctg
    901 tggagtgttt gtgctggttg atgccttctg ggtgtggaat tgtttttccc ggagtggcct
    961 ctgccctctc ccctagcctg tctcagatcc tgggagctgg tgagctgccc cctgcaggtg
    1021 gatcgagtaa ttgcaggggt ttggcaagga ctttgacaga catccccagg ggtgcccggg
    1081 agtgtggggt ccaagccagg agggctgtca gcagtgcacc ttcaccccac agcagagcag
    1141 atttggctgc tctgtcgagc tggatggata ctactttttt tttcctttcc ctctaagtgg
    1201 gggtctcccc cagctactgg agctgtcaga acagtgaagg ctggtaacac atgagttgca
    1261 ctgtgtaagt ttctcgaggc cgggcgcagt ggctcatgcc tgtaatccca gcactttggg
    1321 aggctgaggc aggtggatcg cttgagctca ggagttggag accagcctga ccaacatggt
    1381 gaaaccctgt gtctactaaa aatacaaaga ttagccgggc taggcagtgg gcacctgtaa
    1441 tcacaactgc ttgggaggct gagggaagag aatcgcttga acccaggagg cggaggttgc
    1501 agtgagccga gcttgtgcca ctgcattcca gcctgggcga cagagcaaga ctccgcctca
    1561 aaaaaaaaaa aaaaaagttc ctagaaacag caaaatgtgg agacagaaag cttaccaggg
    1621 attgttgggg aatggggttg ggagagagga ctaactgcag atgaacccaa gggggacttt
    1681 ttaggtgaga gcagtgtcgt gaaaagactg tggtgctgtt tgcgctcaca tttacatttc
    1741 ctaaaattct ttaaacccta cacttggaat ggatgaatta catgacatgc agattgcacc
    1801 ttcataacat aatctttctc ctgggcccct gtctctggct gcctcataaa cgctggtgtt
    1861 tccctcgtgg gcctccctgc atccctgcat ctcctcccgg gtcctgtctg tgagcaatac
    1921 agcgtgacac cctacgctgc cccgtggtcc cgggcttgtc tctccttgcc tccctgttac
    1981 ctttctttct atctcttcct tgccccgtgc actcaacctt gcatccccaa accaaaccta
    2041 ttattcatgg accccaaact tgttcctctt atgtcctgtc cctttgaggg gcaccaccat
    2101 ccacccgcat ggccaagcca gaaaccgtgg tctgctctcc ctccgttaaa tgccattctc
    2161 catcagtgag gcttcttagt catctctggc tgcctggcca ggccctggct gtggcctcct
    2221 ccctggtctt tgtagctctg gatatccctg cagaaagggt ccccactacc aggcctctcc
    2281 atccccagtc tcaggtagtt tttctaaaat gcaaacccca ccctgcaact taccgcccac
    2341 agcccagccc actcttctcc aggcctcgcc tccctccctt ccccctgcac cccacgactt
    2401 ctccagcact gagctgcttc ctgtgcccca cagtggcctg gagtcccctt tgccttaact
    2461 ctttgcccca tagtacagcg gggtctgctc tgattgtagg ggcttcccac atcccccagg
    2521 atggctgccc tctgctgtgg catcactgtg taacaatggc gtgtacacct ctctgtcccc
    2581 accagtgcag ggcccttctc atcgtagggg ctttagctgg ggtttgtgga tcgactgagt
    2641 gaacgaatgt tgtgggaagt cccgtttccc agccgcaccc agggaaattc cacagagcgg
    2701 gcaggggcat cgcatgaggt gctggtgttc acgccagacc acaattaggt gtttaatttt
    2761 taaaaagaaa gttacaacct ttttttttta tttttatttt ttctgattct gcaaataaca
    2821 cctgctctta cagaccatgt gggtgatgtg gaaaagacct gtgaccttct ccatgtccac
    2881 ttctccccac agatctgtac tgcaccctgg aggtggattc ctttgggtat tttgtgaata
    2941 aagcaaagac gcgcgtctac agggacacag ctgagcca
    Serine/threonine-protein kinase B-raf, isoform 1 NP_004324.2 (SEQ ID NO: 188)
    1 maalsggggg gaepgqalfn gdmepeagag agaaassaad paipeevwni kqmikltqeh
    61 iealldkfgg ehnppsiyle ayeeytskld alqqreqqll eslgngtdfs vsssasmdtv
    121 tsssssslsv lpsslsvfqn ptdvarsnpk spqkpivrvf lpnkqrtvvp arcgvtvrds
    181 lkkalmmrgl ipeccavyri qdgekkpigw dtdiswltge elhvevlenv pltthnfvrk
    241 tfftlafcdf crkllfqgfr cqtcgykfhq rcstevplmc vnydqldllf vskffehhpi
    301 pqeeaslaet altsgsspsa pasdsigpqi ltspspsksi pipqpfrpad edhrnqfgqr
    361 drsssapnvh intiepvnid dlirdqgfrg dggsttglsa tppaslpgsl tnvkalqksp
    421 gpqrerksss ssedrnrmkt lgrrdssddw eipdgqitvg qrigsgsfgt vykgkwhgdv
    481 avkmlnvtap tpqqlqafkn evgvlrktrh vnillfmgys tkpqlaivtg wcegsslyhh
    541 lhiietkfem iklidiarqt aqgmdylhak siihrdlksn niflhedltv kigdfglatv
    601 ksrwsgshqf eqlsgsilwm apevirmqdk npysfqsdvy afgivlyelm tgqlpysnin
    661 nrdqiifmvg rgylspdlsk vrsncpkamk rlmaeclkkk rderplfpqi lasiellars
    721 lpkihrsase pslnragfqt edfslyacas pktpiqaggy gafpvh
    Serine/threonine-protein kinase B-raf, isoform 2 NP_001341538.1 (SEQ ID NO:
    189)
    1 maalsggggg gaepgqalfn gdmepeagag agaaassaad paipeevwni kqmikltqeh
    61 iealldkfgg ehnppsiyle ayeeytskld alqqreqqll eslgngtdfs vsssasmdtv
    121 tsssssslsv lpsslsvfqn ptdvarsnpk spqkpivrvf lpnkqrtvvp arcgvtvrds
    181 lkkalmmrgl ipeccavyri qdgekkpigw dtdiswltge elhvevlenv pltthnfvrk
    241 tfftlafcdf crkllfqgfr cqtcgykfhq rcstevplmc vnydqldllf vskffehhpi
    301 pqeeaslaet altsgsspsa pasdsigpqi ltspspsksi pipqpfrpad edhrnqfgqr
    361 drsssapnvh intiepvnid dlirdqgfrg dggsttglsa tppaslpgsl tnvkalqksp
    421 gpqrerksss ssedrnrmkt lgrrdssddw eipdgqitvg qrigsgsfgt vykgkwhgdv
    481 avkmlnvtap tpqqlqafkn evgvlrktrh vnillfmgys tkpqlaivtq wcegsslyhh
    541 lhiietkfem iklidiarqt aqgmdylhak siihrdlksn niflhedltv kigdfglatv
    601 ksrwsgshqf eqlsgsilwm apevirmqdk npysfqsdvy afgivlyelm tgqlpysnin
    661 nrdqiifmvg rgylspdlsk vrsncpkamk rlmaeclkkk rderplfpqi lasiellars
    721 lpkihrsase pslnragfqt edfslyacas pktpiqaggy gefaafk
    Carbonic anhydrase 9 precursor NP_001207.2 (SEQ ID NO: 190)
    1 maplcpspwl pllipapapg ltvqlllsll llvpvhpqrl prmqedsplg ggssgeddpl
    61 geedlpseed spreedppge edlpgeedlp geedlpevkp kseeegslkl edlptveapg
    121 dpqepqnnah rdkegddqsh wryggdppwp rvspacagrf qspvdirpql aafcpalrpl
    181 ellgfqlppl pelrlrnngh svqltlppgl emalgpgrey ralqlhlhwg aagrpgseht
    241 veghrfpaei hvvhlstafa rvdealgrpg glavlaafle egpeensaye qllsrleeia
    301 eegsetqvpg ldisallpsd fsryfqyegs lttppcaqgv iwtvfnqtvm lsakqlhtls
    361 dtlwgpgdsr lqlnfratqp lngrvieasf pagvdsspra aepvqlnscl aagdilalvf
    421 gllfavtsva flvqmrrqhr rgtkggvsyr paevaetga
    G/mitotic-specific cyclin-B1, isoform 1 NP_114172.1 (SEQ ID NO: 191)
    1 malrvtrnsk inaenkakin magakrvpta paatskpglr prtalgdign kvseqlqakm
    61 pmkkeakpsa tgkvidkklp kplekvpmlv pvpvsepvpe pepepepepv keeklspepi
    121 lvdtaspspm etsgcapaee dlcqafsdvi lavndvdaed gadpnlcsey vkdiyaylrq
    181 leeeqavrpk yllgrevtgn mrailidwlv qvqmkfrllq etmymtvsii drfmqnncvp
    241 kkmlqlvgvt amfiaskyee myppeigdfa fvtdntytkh girqmemkil ralnfglgrp
    301 lplhflrras kigevdveqh tlakylmelt mldydmvhfp psqiaagafc lalkildnge
    361 wtptlqhyls yteesllpvm qhlaknvvmv nqgltkhmtv knkyatskha kistlpqlns
    421 alvqdlakav akv
    G/mitotic-specific cyclin-B1, isoform 2 NP_001341773.1 (SEQ ID NO: 192)
    1 malrvtrnsk inaenkakin magakrvpta paatskpglr prtalgdign kvseqlqakm
    61 pmkkeakpsa tgkvidkklp kplekvpmlv pvpvsepvpe pepepepepv keeklspepi
    121 lvdtaspspm etsgcapaee dlcqafsdvi lavndvdaed gadpnlcsey vkdiyaylrq
    181 leeeqavrpk yllgrevtgn mrailidwlv qvqmkfrllq etmymtvsii drfmqnncvp
    241 kkmlqlvgvt amfiaskyee myppeigdfa fvtdntytkh qirqmemkil ralnfglgrp
    301 lplhflrras kigevdveqh tlakylmelt mldydmvhfp psqiaagafc lalkildnge
    361 wtvknkyats khakistlpq lnsalvqdla kavakv
    G/mitotic-specific cyclin-B1, isoform 3 NP_001341774.1 (SEQ ID NO: 193)
    1 malrvtrnsk inaenkakin magakrvpta paatskpglr prtalgdign kvseqlqakm
    61 pmkkeakpsa tgkvidkklp kplekvpmlv pvpvsepvpe pepepepepv keeklspepi
    121 lvdtaspspm etsgcapaee dlcqafsdvi lavndvdaed gadpnlcsey vkdiyaylrq
    181 lenncvpkkm lqlvgvtamf iaskyeemyp peigdfafvt dntytkhqir qmemkilral
    241 nfglgrplpl hflrraskig evdveqhtla kylmeltmld ydmvhfppsq iaagafclal
    301 kildngewtp tlqhylsyte esllpvmghl aknvvmvnqg ltkhmtvknk yatskhakis
    361 tlpqlnsalv qdlakavakv
    CD276, isoform a precursor NP_001019907.1 (SEQ ID NO: 194)
    1 mlrrrgspgm gvhvgaalga lwfcltgale vqvpedpvva lvgtdaticc sfspepgfsl
    61 aqlnliwqlt dtkqlvhsfa egqdqgsaya nrtalfpdll aqgnaslrlq rvrvadegsf
    121 tcfvsirdfg saayslqvaa pyskpsmtle pnkdlrpgdt vtitcssyqg ypeaevfwqd
    181 gqgvpltgnv ttsqmaneqg lfdvhsilrv vlgangtysc lvrnpvlqqd ahssvtitpq
    241 rsptgavevq vpedpvvalv gtdatlrcsf spepgfslaq lnliwqltdt kqlvhsfteg
    301 rdqgsayanr talfpdllaq gnaslrlqrv rvadegsftc fvsirdfgsa ayslqvaapy
    361 skpsmtlepn kdlrpgdtvt itcssyrgyp eaevfwqdgq gvpltgnvtt sqmaneqglf
    421 dvhsvlrvvl gangtysclv rnpvlqqdah gsvtitgqpm tfppealwvt vglsvclial
    481 lvalafvcwr kikqsceeen agaedqdgeg egsktalqpl khsdskeddg qeia
    CD276, isoform b precursor NP_001316557.1, NP_079516.1 (SEQ ID NO: 195)
    1 mlrrrgspgm gvhvgaalga lwfcltgale vqvpedpvva lvgtdatlcc sfspepgfsl
    61 aqlnliwqlt dtkqlvhsfa egqdqgsaya nrtalfpdll aqgnaslrlq rvrvadegsf
    121 tcfvsirdfg saayslqvaa pyskpsmtle pnkdlrpgdt vtitcssyrg ypeaevfwqd
    181 gqgvpltgnv ttsqmaneqg lfdvhsvlrv vlgangtysc lvrnpvlqqd ahgsvtitgq
    241 pmtfppealw vtvglsvcli allvalafvc wrkikqscee enagaedqdg egegsktalq
    301 plkhsdsked dgqeia
    CD276, isoform c NP_001316558.1 (SEQ ID NO: 196)
    1 mtlepnkdlr pgdtvtitcs syqgypeaev fwqdgqgvpl tgnvttsqma neqglfdvhs
    61 ilrvvlgang tysclvrnpv lqqdahssvt itpqrsptga vevqvpedpv valvgtdatl
    121 rcsfspepgf slaqlnliwq ltdtkqlvhs ftegrdqgsa yanrtalfpd llaqgnaslr
    181 lqrvrvadeg sftcfvsird fgsaayslqv aapyskpsmt lepnkdlrpg dtvtitcssy
    241 rgypeaevfw qdgqgvpltg nvttsqmane qglfdvhsvl rvvlgangty sclvrnpvlq
    301 qdahgsvtit gqpmtfppea lwvtvglsvc liallvalaf vcwrkikqsc eeenagaedq
    361 dgegegskta lqplkhsdsk eddgqeia
    Carcinoembryonic antigen-related cell adhesion molecule 3, isoform 1
    precursor NP_001806.2 (SEQ ID NO: 197)
    1 mgppsasphr ecipwqglll tasllnfwnp pttaklties mplsvaegke vlllvhnlpq
    61 hlfgyswykg ervdgnsliv gyvigtqqat pgaaysgret iytnaslliq nvtqndigfy
    121 tlqviksdlv neeatgqfhv yqenapglpv gavagivtgv lvgvalvaal vcflllaktg
    181 rtsiqrdlke qqpqalapgr gpshssafsm splstaqapl pnprtaasiy eellkhdtni
    241 ycrmdhkaev as
    Carcinoembryonic antigen-related cell adhesion molecule 3, isoform 2
    precursor NP_001264092.1 (SEQ ID NO: 198)
    1 mgppsasphr ecipwqglll tasllnfwnp pttaklties mplsvaegke vlllvhnlpq
    61 hlfgyswykg ervdgnsliv gyvigtqqat pgaaysgret iytnaslliq nvtqndigfy
    121 tlqviksdlv neeatgqfhv yqenapglpv gavagivtgv lvgvalvaal vcflllaktg
    181 rpwslpqlcl ldvpslhcpg pptqpqdssf hl
    Carcinoembryonic antigen-related cell adhesion molecule 5, isoform 1
    preprotein NP_001278413.1, NP_004354.3 (SEQ ID NO: 199)
    1 mespsapphr wcipwqrlll taslltfwnp pttaklties tpfnvaegke vlllvhnlpq
    61 hlfgyswykg ervdgnrqii gyvigtqqat pgpaysgrei iypnaslliq niiqndtgfy
    121 tlhviksdlv neeatgqfrv ypelpkpsis snnskpvedk davaftcepe tqdatylwwv
    181 nnqslpvspr lqlsngnrtl tlfnvtrndt asykcetqnp vsarrsdsvi lnvlygpdap
    241 tisplntsyr sgenlnlsch aasnppaqys wfvngtfqqs tqelfipnit vnnsgsytcq
    301 ahnsdtglnr ttvttitvya eppkpfitsn nsnpvededa valtcepeiq nttylwwvnn
    361 qslpvsprlq lsndnrtltl lsvtrndvgp yecgiqnels vdhsdpviln vlygpddpti
    421 spsytyyrpg vnlslschaa snppaqyswl idgniqghtq elfisnitek nsglytcqan
    481 nsasghsrtt vktitvsael pkpsissnns kpvedkdava ftcepeaqnt tylwwvngqs
    541 lpvsprlqls ngnrtltlfn vtrndarayv cgiqnsvsan rsdpvtldvl ygpdtpiisp
    601 pdssylsgan lnlschsasn pspqyswrin gipqqhtqvl fiakitpnnn gtyacfvsnl
    661 atgrnnsivk sitvsasgts pglsagatvg imigvlvgva li
    Carcinoembryonic antigen-related cell adhesion molecule 5, isoform 2
    preprotein NP_001295327.1 (SEQ ID NO: 200)
    1 mespsapphr wcipwqrlll taslltfwnp pttaklties tpfnvaegke vlllvhnlpq
    61 hlfgyswykg ervdgnrqii gyvigtqqat pgpaysgrei iypnaslliq niiqndtgfy
    121 tlhviksdlv neeatgqfrv ypelpkpsis snnskpvedk davaftcepe tqdatylwwv
    181 nnqslpvspr lqlsngnrtl tlfnvtrndt asykcetqnp vsarrsdsvi lnvlygpdap
    241 tispintsyr sgenlnlsch aasnppaqys wfvngtfqqs tqelfipnit vnnsgsytcq
    301 ahnsdtglnr ttvttitvye ppkpfitsnn snpvededav altcepeiqn ttylwwvnnq
    361 slpvsprlql sndnrtltll svtrndvgpy ecgiqnelsv dhsdpvilnv lygpddptis
    421 psytyyrpgv nlslschaas nppaqyswli dgniqqhtqe lfisnitekn sglytcqann
    481 sasghsrttv ktitvsaelp kpsissnnsk pvedkdavaf tcepeaqntt ylwwvngqsl
    541 pvsprlqlsn gnrtltlfnv trndarayvc giqnsysanr sdpvtldvly gpdtpiispp
    601 dssylsganl nlschsasnp spqyswring ipqqhtqvlf iakitpnnng tyacfvsnla
    661 tgrnnsivks itvsasgtsp glsagatvgi migvlvgval i
    Baculoviral IAP repeat containing 2, isoform 1 NP_001157.1, NP_001243092.1
    (SEQ ID NO: 201)
    1 mhktasqrlf pgpsyqniks imedstilsd wtnsnkqkmk ydfscelyrm stystfpagv
    61 pvserslara gfyytgvndk vkcfccglml dnwklgdspi qkhkqlypsc sfiqnlvsas
    121 lgstskntsp mrnsfahsls ptlehsslfs gsysslspnp lnsravedis ssrtnpysya
    181 msteearflt yhmwpltfls pselaragfy yigpgdrvac facggklsnw epkddamseh
    241 rrhfpncpfl ensletlrfs isnlsmqtha armrtfmywp ssvpvqpeql asagfyyvgr
    301 nddvkcfccd gglrcwesgd dpwvehakwf prceflirmk gqefvdeiqg ryphlleqll
    361 stsdttgeen adppiihfgp gesssedavm mntpvvksal emgfnrdlvk qtvqskiltt
    421 genyktvndi vsallnaede kreeekekqa eemasddlsl irknrmalfq qltcvlpild
    481 nllkanvink qehdiikqkt qiplqareli dtilvkgnaa anifknclke idstlyknlf
    541 vdknmkyipt edvsglslee qlrrlqeert ckvcmdkevs vvfipcghlv vcqecapslr
    601 kcpicrgiik gtvrtfls
    Baculoviral IAP repeat containing 2, isoform 2 NP_001243095.1 (SEQ ID NO:
    202)
    1 mstystfpag vpvserslar agfyytgvnd kvkcfccglm ldnwklgdsp iqkhkqlyps
    61 csfiqnlvsa slgstsknts pmrnsfahsl sptlehsslf sgsysslspn plnsravedi
    121 sssrtnpysy amsteearfl tyhmwpltfl spselaragf yyigpgdrva cfacggklsn
    181 wepkddamse hrrhfpncpf lensletlrf sisnlsmqth aarmrtfmyw pssvpvqpeq
    241 lasagfyyvg rnddvkcfcc dgglrcwesg ddpwvehakw fprceflirm kgqefvdeiq
    301 gryphlleql lstsdttgee nadppiihfg pgesssedav mmntpvvksa lemgfnrdlv
    361 kqtvqskilt tgenyktvnd ivsallnaed ekreeekekq aeemasddls lirknrmalf
    421 qqltcvlpil dnllkanvin kqehdiikqk tqiplqarel idtilvkgna aanifknclk
    481 eidstlyknl fvdknmkyip tedvsglsle eqlrrlqeer tckvcmdkev svvfipcghl
    541 vvcqecapsl rkcpicrgii kgtvrtfls
    Chondrosarcoma-associated gene 2/3 protein, isoform X1 XP_006724920.1 (SEQ ID
    NO: 203)
    1 mwmgliqlve gvkrkdqgfl ekefyhktni kmrceflacw paftvlgeaw rdqvdwsrll
    61 rdtglvkmsr kprassplsn nhpptpkrrg sgrhpinpgp ealskfprqp grekgpikev
    121 pgtkgsp
    Chondrosarcoma-associated gene 2/3 protein, isoform X2 XP_016885512.1 (SEQ ID
    NO: 204)
    1 mwmgliqlve gvkrkdqgfl ekefyhktni kmrceflacw paftvlgeaw rdqvdwsrll
    61 rdtglvkmsr kprassplsn nhpptpkrfp rqpgrekgpi kevpgtkgsp
    Chondroitin sulfate proteoglycan 4 precursor NP_001888.2 (SEQ ID NO: 205)
    1 mqsgprpplp apglalaltl tmlarlasaa sffgenhlev pvataltdid lqlqfstsqp
    61 eallllaagp adhlllqlys grlqvrlvlg qeelrlqtpa etllsdsiph tvvltvvegw
    121 atlsvdgfln assavpgapl evpyglfvgg tgtlglpylr gtsrplrgcl haatlngrsl
    181 lrpltpdvhe gcaeefsasd dvalgfsgph slaafpawgt qdegtleftl ttqsrqapla
    241 fqaggrrgdf iyvdifeghl ravvekgqgt vllhnsvpva dgqphevsvh inahrleisv
    301 dqypthtsnr gvlsyleprg slllggldae asrhlqehrl gltpeatnas llgcmedlsv
    361 ngqrrglrea lltrnmaagc rleeeeyedd ayghyeafst lapeawpame lpepcvpepg
    421 lppvfanftq lltisplvva eggtawlewr hvqptldlme aelrksqvlf svtrgarhge
    481 leldipgaqa rkmftlldvv nrkarfihdg sedtsdqlvl evsvtarvpm psclrrgqty
    541 llpiqvnpvn dpphiifphg slmvilehtq kplgpevfqa ydpdsacegl tfqvlgtssg
    601 lpverrdqpg epatefscre leagslvyvh rggpaqdltf rvsdglqasp patlkvvair
    661 paiqihrstg lrlaqgsamp ilpanlsvet navgqdvsvl frvtgalqfg elqkqgaggv
    721 egaewwatqa fhqrdveqgr vrylstdpqh haydtvenla levqvgqeil snlsfpvtiq
    781 ratvwmlrle plhtqntqqe tlttahleat leeagpsppt fhyevvqapr kgnlqlqgtr
    841 lsdgqgftqd diqagrvtyg ataraseave dtfrfrvtap pyfsplytfp ihiggdpdap
    901 vltnvllvvp eggegvlsad hlfvkslnsa sylyevmerp rhgrlawrgt qdkttmvtsf
    961 tnedllrgrl vyqhddsett eddipfvatr qgessgdmaw eevrgvfrva iqpvndhapv
    1021 qtisrifhva rggrrllttd dvafsdadsg fadaqlvltr kdllfgsiva vdeptrpiyr
    1081 ftqedlrkrr vlfvhsgadr gwiqlqvsdg qhqatallev qasepylrva ngsslvvpqg
    1141 gqgtidtavl hldtnldirs gdevhyhvta gprwgqlvra gqpatafsqq dlldgavlys
    1201 hngslsprdt mafsveagpv htdatlqvti alegplaplk lvrhkkiyvf qgeaaeirrd
    1261 qleaaqeavp padivfsvks ppsagylvmv srgaladepp sldpvqsfsq eavdtgrvly
    1321 lhsrpeawsd afsldvasgl gaplegvlve levlpaaipl eaqnfsvpeg gsltlappll
    1381 rvsgpyfptl lglslqvlep pqhgalqked gpqartlsaf swrmveeqli ryvhdgsetl
    1441 tdsfvlmana semdrqshpv aftvtvlpvn dqppilttnt glqmwegata pipaealrst
    1501 dgdsgsedlv ytieqpsngr vvlrgapgte vrsftqaqld gglvlfshrg tldggfrfrl
    1561 sdgehtspgh ffrvtaqkqv llslkgsqtl tvcpgsvqpl ssqtlrasss agtdpqllly
    1621 rvvrgpqlgr lfhaqqdstg ealvnftqae vyagnilyeh emppepfwea hdtlelqlss
    1681 ppardvaatl avavsfeaac pqrpshlwkn kglwvpegqr aritvaalda snllasvpsp
    1741 qrsehdvlfq vtqfpsrgql lvseeplhag qphflqsqla agqlvyahgg ggtqqdgfhf
    1801 rahlqgpaga svagpqtsea faitvrdvne rppqpqasvp lrltrgsrap israqlsvvd
    1861 pdsapgeiey evqraphngf lslvggglgp vtrftqadvd sgrlafvang ssvagifqls
    1921 msdgaspplp mslavdilps aievqlrapl evpqalgrss lsqqqlrvvs dreepeaayr
    1981 liqgpqyghl lvggrptsaf sqfqidqgev vfaftnfsss hdhfrvlala rgvnasavvn
    2041 vtvrallhvw aggpwpqgat lrldptvlda gelanrtgsv prfrllegpr hgrvvrvpra
    2101 rtepggsqlv eqftqqdled grlglevgrp egrapgpagd sltlelwaqg vppavasldf
    2161 atepynaarp ysvallsvpe aarteagkpe sstptgepgp masspepava kggflsflea
    2221 nmfsviipmc lvllllalil pllfylrkrn ktgkhdvqvl takprnglag dtetfrkvep
    2281 gqaipltavp gqgpppggqp dpellqfcrt pnpalkngqy wv
    Cancer/testis antigen 2 isoform LAGE-1a NP_758965.2 (SEQ ID NO: 206)
    1 mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgaga arasgprgga
    61 prgphggaas aqdgrcpcga rrpdsrllel hitmpfsspm eaelvrrils rdaaplprpg
    121 avlkdftvsg nllfirltaa dhrqlqlsis sclqqlsllm witqcflpvf laqapsgqrr
    Cancer/testis antigen 2 isoform LAGE-1b NP_066274.2 (SEQ ID NO: 207)
    1 mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgaga arasgprgga
    61 prgphggaas aqdgrcpcga rrpdsrllel hitmpfsspm eaelvrrils rdaaplprpg
    121 avlkdftvsg nllfmsvrdq dregagrmrv vgwglgsasp egqkardlrt pkhkvseqrp
    181 gtpgppppeg aqgdgcrgva fnvmfsaphi
    Transcriptional repressor CTCFL, isoform 1 NP_001255969.1, NP_001255970.1,
    NP_542185.2 (SEQ ID NO: 208)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveasklkrh
    361 vrshtgerpf qccqcsyasr dtyklkrhmr thsgekpyec hichtrftqs gtmkihilqk
    421 hgenvpkyqc phcatiiark sdlrvhmrnl haysaaelkc rycsavfher yaliqhqkth
    481 knekrfkckh csyackqerh mtahirthtg ekpftclscn kcfrqkqlln ahfrkyhdan
    541 fiptvykcsk cgkgfsrwin lhrhsekcgs geaksaasgk grrtrkrkqt ilkeatkgqk
    601 eaakgwkeaa ngdeaaaeea sttkgeqfpg emfpvacret tarvkeevde gvtcemllnt
    661 mdk
    Transcriptional repressor CTCFL, isoform 2 NP_001255971.1 (SEQ ID NO: 209)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveasklkrh
    361 vrshtgerpf qccqcsyasr dtyklkrhmr thsgekpyec hichtrftqs gtmkihilqk
    421 hgenvpkyqc phcatiiark sdlrvhmrnl haysaaelkc rycsavfher yaliqhqkth
    481 knekrfkckh csyackqerh mtahirthtg ekpftclscn kcfrqkqlln ahfrkyhdan
    541 fiptvykcsk cgkgfsrwin lhrhsekcgs geaksaasgk grrtrkrkqt ilkeatkgqk
    601 eaakgwkeaa ngdaaaeeas ttkgeqfpge mfpvacrett arvkeevdeg vtcemllntm
    661 dk
    Transcriptional repressor CTCFL, isoform 3 NP_001255972.1 (SEQ ID NO: 210)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveasklkrh
    361 vrshtgerpf qccqcsyasr dtyklkrhmr thsgekpyec hichtrftqs gtmkihilqk
    421 hgenvpkyqc phcatiiark sdlrvhmrnl haysaaelkc rycsavfher yaliqhqkth
    481 knekrfkckh csyackqerh mtahirthtg ekpftclscn kcfrqkqlln ahfrkyhdan
    541 fiptvykcsk cgkgfsrwin lhrhsekcgs geaksaasgk grrtrkrkqt ilkeatkgqk
    601 eaakgwkeaa ngdeaaaeea sttkgeqfpg emfpvacret tarvkeevde gvtcemllnt
    661 mdnsagctgr mmlvsawllg rpqetynqgr rrrgsrrvtw
    Transcriptional repressor CTCFL, isoform 4 NP_001255973.1 (SEQ ID NO: 211)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveasklkrh
    361 vrshtgerpf qccqcsyasr dtyklkrhmr thsgekpyec hichtrftqs gtmkihilqk
    421 hgenvpkyqc phcatiiark sdlrvhmrnl haysaaelkc rycsavfher yaliqhqkth
    481 knekrfkckh csyackqerh mtahirthtg ekpftclscn kcfrqkqlln ahfrkyhdan
    541 fiptvykcsk cgkgfsrwin lhrhsekcgs geaksaasgk grrtrkrkqt ilkeatkgqk
    601 eaakgwkeaa ngdgvisahr nlcllgssds hasysgagit darhhawliv llflvemgfy
    661 hvshs
    Transcriptional repressor CTCFL, isoform 5 NP_001255974.1 (SEQ ID NO: 212)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveasklkrh
    361 vrshtgerpf qccqcsyasr dtyklkrhmr thsgekpyec hichtrftqs gtmkihilqk
    421 hgenvpkyqc phcatiiark sdlrvhmrnl haysaaelkc rycsavfher yaliqhqkth
    481 knekrfkckh csyackqerh mtahirthtg ekpftclscn kcfrqkqlln ahfrkyhdan
    541 fiptvykcsk cgkgfsrwil wvgnsevael ggpgsgpllr lqsgcppglh hpkaglgped
    601 plpgqlrhtt agtglssllq gplcraa
    Transcriptional repressor CTCFL, isoform 6 NP_001255975.1 (SEQ ID NO: 213)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveasklkrh
    361 vrshtgerpf qccqcsyasr dtyklkrhmr thsgvhmrnl haysaaelkc rycsavfher
    421 yaliqhqkth knekrfkckh csyackqerh mtahirthtg ekpftclscn kcfrqkqlln
    481 ahfrkyhdan fiptvykcsk cgkgfsrwin lhrhsekcgs geaksaasgk grrtrkrkqt
    541 ilkeatkgqk eaakgwkeaa ngdeaaaeea sttkgeqfpg emfpvacret tarvkeevde
    601 gvtcemllnt mdk
    Transcriptional repressor CTCFL, isoform 7 NP_001255976.1 (SEQ ID NO: 214)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveasklkrh
    361 vrshtgerpf qccqcsyasr dtyklkrhmr thsgekpyec hichtrftqs gtmkihilqk
    421 hgenvpkyqc phcatiiark sdlrvhmrnl haysaaelkc rycsavfher yaliqhqkth
    481 knekrfkckh csyackqerh mtahirthtg ekpftclscn kcfrqkqlln ahfrkyhdan
    541 fiptvykcsk cgkgfsrwit skwsglkpqt fit
    Transcriptional repressor CTCFL, isoform 8 NP_001255977.1 (SEQ ID NO: 215)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya sveerhmtah
    361 irthtgekpf tclscnkcfr qkqllnahfr kyhdanfipt vykcskcgkg fsrwilwvgn
    421 sevaelggpg sgpllrlqsg cppglhhpka glgpedplpg qlrhttagtg lssllqgplc
    481 raa
    Transcriptional repressor CTCFL, isoform 9 NP_001255978.1 (SEQ ID NO: 216)
    1 msgdersdei vltvsnsnve eqedqptagq adaekakstk nqrktkgakg tfhcdvcmft
    61 ssrmssfnrh mkthtsekph lchlclktfr tvtllrnhvn thtgtrpykc ndcnmafvts
    121 gelvrhrryk hthekpfkcs mckyasveas klkrhvrsht gerpfqccqc syasrdtykl
    181 krhmrthsge kpyechicht rftqsgtmki hilqkhgenv pkyqcphcat iiarksdlrv
    241 hmrnlhaysa aelkcrycsa vfheryaliq hqkthknekr fkckhcsyac kqerhmtahi
    301 rthtgekpft clscnkcfrq kqllnahfrk yhdanfiptv ykcskcgkgf srwinlhrhs
    361 ekcgsgeaks aasgkgrrtr krkqtilkea tkgqkeaakg wkeaangdgv isahrnlcll
    421 gssdshasvs gagitdarhh awlivllflv emgfyhvshs
    Transcriptional repressor CTCFL, isoform 10 NP_001255979.1 (SEQ ID NO: 217)
    1 msgdersdei vltvsnsnve eqedqptagq adaekakstk nqrktkgakg tfhcdvcmft
    61 ssrmssfnrh mkthtsekph lchlclktfr tvtllrnhvn thtgtrpykc ndcnmafvts
    121 gelvrhrryk hthekpfkcs mckyasveas klkrhvrsht gerpfqccqc syasrdtykl
    181 krhmrthsge kpyechicht rftqsgtmki hilqkhgenv pkyqcphcat iiarksdlrv
    241 hmrnlhaysa aelkcrycsa vfheryaliq hqkthknekr fkckhcsyac kqerhmtahi
    301 rthtgekpft clscnkcfrq kqllnahfrk yhdanfiptv ykcskcgkgf srwilwvgns
    361 evaelggpgs gpllrlqsgc ppglhhpkag lgpedplpgq lrhttagtgl ssllqgplcr
    421 aa
    Transcriptional repressor CTCFL, isoform 11 NP_001255980.1, NP_001255981.1
    (SEQ ID NO: 218)
    1 maateisvls eqftkikele lmpekglkee ekdgvcrekd hrspseleae rtsgafqdsv
    61 leeevelvla pseesekyil tlqtvhftse avelqdmsll siqqqegvqv vvqqpgpgll
    121 wleegprqsl qqcvaisiqq elyspqemev lqfhaleenv mvasedskla vslaettgli
    181 kleeeqeknq llaertkeql ffvetmsgde rsdeivltvs nsnveeqedq ptagqadaek
    241 akstknqrkt kgakgtfhcd vcmftssrms sfnrhmktht sekphlchlc lktfrtvtll
    301 rnhvnthtgt rpykcndcnm afvtsgelvr hrrykhthek pfkcsmckya svevkpfldl
    361 klhgilveaa vqvtpsvtns ricykqafyy sykiyagnnm hsll
    Transcriptional repressor CTCFL, isoform 12 NP_001255983.1 (SEQ ID NO: 219)
    1 mftssrmssf nrhmkthtse kphlchlclk tfrtvtllrn hvnthtgtrp ykcndcnmaf
    61 vtsgelvrhr rykhthekpf kcsmckyasv easklkrhvr shtgerpfqc cqcsyasrdt
    121 yklkrhmrth sgekpyechi chtrftqsgt mkihilqkhg envpkyqcph catiiarksd
    181 lrvhmrnlha ysaaelkcry csavfherya liqhqkthkn ekrfkckhcs yackqerhmt
    241 ahirthtgek pftclscnkc frqkqllnah frkyhdanfi ptvykcskcg kgfsrwinlh
    301 rhsekcgsge aksaasgkgr rtrkrkqtil keatkgqkea akgwkeaang dgvisahrnl
    361 cllgssdsha sysgagitda rhhawlivll flvemgfyhv shs
    Transcriptional repressor CTCFL, isoform 13 NP_001255984.1 (SEQ ID NO: 220)
    1 mftssrmssf nrhmkthtse kphlchlclk tfrtvtllrn hvnthtgtrp ykcndcnmaf
    61 vtsgelvrhr rykhthekpf kcsmckyasv easklkrhvr shtgerpfqc cqcsyasrdt
    121 yklkrhmrth sgekpyechi chtrftqsgt mkihilqkhg envpkyqcph catiiarksd
    181 lrvhmrnlha ysaaelkcry csavfherya liqhqkthkn ekrfkckhcs yackqerhmt
    241 ahirthtgek pftclscnkc frqkqllnah frkyhdanfi ptvykcskcg kgfsrwvly
    Cytochrome P450 1B1 NP_000095.2 (SEQ ID NO: 221)
    1 mgtslspndp wplnplsiqq ttlllllsvl atvhvgqrll rqrrrqlrsa ppgpfawpli
    61 gnaaavgqaa hlsfarlarr ygdvfqirlg scpivvlnge raihqalvqq gsafadrpaf
    121 asfrvvsggr smafghyseh wkvqrraahs mmrnfftrqp rsrqvleghv lsearelval
    181 lvrgsadgaf ldprpltvva vanvmsavcf gcryshddpe frellshnee fgrtvgagsl
    241 vdvmpwlqyf pnpvrtvfre feqlnrnfsn fildkflrhc eslrpgaapr dmmdafilsa
    301 ekkaagdshg ggarldlenv patitdifga sqdtlstalq wllllftryp dvqtrvqael
    361 dqvvgrdrlp cmgdqpnlpy vlaflyeamr fssfvpvtip hattantsvl gyhipkdtvv
    421 fvnqwsvnhd plkwpnpenf dparfldkdg linkdltsrv mifsvgkrrc igeelskmql
    481 flfisilahq cdfranpnep akmnfsyglt ikpksfkvnv tlresmelld savqnlqake
    541 tcq
    Epidermal growth factor receptor, isoform a precursor NP_005219.2 (SEQ ID NO:
    222)
    1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev
    61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala
    121 vlsnydankt glkelpmrnl qeilhgavrf snnpalcnve siqwrdivss dflsnmsmdf
    181 qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcs grcrgkspsd cchnqcaagc
    241 tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvn pegkysfgat cvkkcprnyv
    301 vtdhgscvra cgadsyemee dgvrkckkce gpcrkvcngi gigefkdsls inatnikhfk
    361 nctsisgdlh ilpvafrgds fthtppldpq eldilktvke itgflliqaw penrtdlhaf
    421 enleiirgrt kqhgqfslav vslnitslgl rslkeisdgd viisgnknlc yantinwkkl
    481 fgtsgqktki isnrgensck atgqvchalc spegcwgpep rdcvscrnvs rgrecvdkcn
    541 llegeprefv enseciqchp eclpqamnit ctgrgpdnci qcahyidgph cvktcpagvm
    601 genntlvwky adaghvchlc hpnctygctg pglegcptng pkipsiatgm vgalllllvv
    661 algiglfmrr rhivrkrtlr rllqerelve pltpsgeapn qallrilket efkkikvlgs
    721 gafgtvykgl wipegekvki pvaikelrea tspkankeil deayvmasvd nphvcrllgi
    781 cltstvqlit qlmpfgclld yvrehkdnig sqyllnwcvq iakgmnyled rrlvhrdlaa
    841 rnvlvktpqh vkitdfglak llgaeekeyh aeggkvpikw malesilhri ythqsdvwsy
    901 gvtvwelmtf gskpydgipa seissilekg erlpqppict idvymimvkc wmidadsrpk
    961 freliiefsk mardpqrylv iqgdermhlp sptdsnfyra lmdeedmddv vdadeylipq
    1021 qgffsspsts rtpllsslsa tsnnstvaci drnglqscpi kedsflqrys sdptgalted
    1081 siddtflpvp eyinqsvpkr pagsvqnpvy hnqplnpaps rdphyqdphs tavgnpeyln
    1141 tvqptcvnst fdspahwaqk gshqisldnp dyqqdffpke akpngifkgs taenaeylry
    1201 apqssefiga
    Epidermal growth factor receptor, isoform b precursor NP_958439.1 (SEQ ID NO:
    223)
    1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev
    61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala
    121 vlsnydankt glkelpmrnl qeilhgavrf snnpalcnve siqwrdivss dflsnmsmdf
    181 qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcs grcrgkspsd cchnqcaagc
    241 tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvn pegkysfgat cvkkcprnyv
    301 vtdhgscvra cgadsyemee dgvrkckkce gpcrkvcngi gigefkdsls inatnikhfk
    361 nctsisgdlh ilpvafrgds fthtppldpq eldilktvke itgflliqaw penrtdlhaf
    421 enleiirgrt kqhgqfslav vslnitslgl rslkeisdgd viisgnknlc yantinwkkl
    481 fgtsgqktki isnrgensck atgqvchalc spegcwgpep rdcvscrnvs rgrecvdkcn
    541 llegeprefv enseciqchp eclpqamnit ctgrgpdnci qcahyidgph cvktcpagvm
    601 genntlvwky adaghvchlc hpnctygs
    Epidermal growth factor receptor, isoform c precursor NP_958440.1 (SEQ ID NO:
    224)
    1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev
    61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala
    121 vlsnydankt glkelpmrnl qeilhgavrf snnpalcnve siqwrdivss dflsnmsmdf
    181 qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcs grcrgkspsd cchnqcaagc
    241 tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvn pegkysfgat cvkkcprnyv
    301 vtdhgscvra cgadsyemee dgvrkckkce gpcrkvcngi gigefkdsls inatnikhfk
    361 nctsisgdlh ilpvafrgds fthtppldpq eldilktvke itgls
    Epidermal growth factor receptor, isoform d precursor NP_958441.1 (SEQ ID NO:
    225)
    1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev
    61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala
    121 vlsnydankt glkelpmrnl qeilhgavrf snnpalcnve siqwrdivss dflsnmsmdf
    181 qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcs grcrgkspsd cchnqcaagc
    241 tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvn pegkysfgat cvkkcprnyv
    301 vtdhgscvra cgadsyemee dgvrkckkce gpcrkvcngi gigefkdsls inatnikhfk
    361 nctsisgdlh ilpvafrgds fthtppldpq eldilktvke itgflliqaw penrtdlhaf
    421 enleiirgrt kqhgqfslav vslnitslgl rslkeisdgd viisgnknlc yantinwkkl
    481 fgtsgqktki isnrgensck atgqvchalc spegcwgpep rdcvscrnvs rgrecvdkcn
    541 llegeprefv enseciqchp eclpqamnit ctgrgpdnci qcahyidgph cvktcpagvm
    601 genntlvwky adaghvchlc hpnctygpgn eslkamlfcl fklsscnqsn dgsyshqsgs
    661 paaqesclgw ipsllpsefq lgwggcshlh awpsasviit assch
    Epidermal growth factor receptor, isoform e precursor NP_001333826.1 (SEQ ID
    NO: 226)
    1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev
    61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala
    121 vlsnydankt glkelpmrnl qgqkcdpscp ngscwgagee ncqkltkiic aqqcsgrcrg
    181 kspsdcchnq caagctgpre sdclvcrkfr deatckdtcp plmlynptty qmdvnpegky
    241 sfgatcvkkc prnyvvtdhg scvracgads yemeedgvrk ckkcegpcrk vcngigigef
    301 kdslsinatn ikhfknctsi sgdlhilpva frgdsfthtp pldpqeldil ktvkeitgfl
    361 liqawpenrt dlhafenlei irgrtkqhgq fslavvslni tslglrslke isdgdviisg
    421 nknlcyanti nwkklfgtsg qktkiisnrg ensckatgqv chalcspegc wgpeprdcvs
    481 crnvsrgrec vdkcnllege prefvensec iqchpeclpq amnitctgrg pdnciqcahy
    541 idgphcvktc pagvmgennt lvwkyadagh vchlchpnct ygctgpgleg cptngpkips
    601 iatgmvgall lllvvalgig lfmrrrhivr krtlrrllqe relvepltps geapnqallr
    661 ilketefkki kvlgsgafgt vykglwipeg ekvkipvaik elreatspka nkeildeayv
    721 masvdnphvc rllgicltst vqlitqlmpf gclldyvreh kdnigsqyll nwcvqiakgm
    781 nyledrrlvh rdlaarnvlv ktpqhvkitd fglakllgae ekeyhaeggk vpikwmales
    841 ilhriythqs dvwsygvtvw elmtfgskpy dgipaseiss ilekgerlpq ppictidvym
    901 imvkcwmida dsrpkfreli iefskmardp qrylviqgde rmhlpsptds nfyralmdee
    961 dmddvvdade ylipqqgffs spstsrtpll sslsatsnns tvacidrngl qscpikedsf
    1021 lqryssdptg altedsiddt flpvpgewlv wkqscsstss thsaaaslqc psqvlppasp
    1081 egetvadlqt q
    Epidermal growth factor receptor, isoform f precursor NP_001333827.1 (SEQ ID
    NO: 227)
    1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev
    61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala
    121 vlsnydankt glkelpmrnl qeilhgavrf snnpalcnve siqwrdivss dflsnmsmdf
    181 qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcs grcrgkspsd cchnqcaagc
    241 tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvn pegkysfgat cvkkcprnyv
    301 vtdhgscvra cgadsyemee dgvrkckkce gpcrkvcngi gigefkdsls inatnikhfk
    361 nctsisgdlh ilpvafrgds fthtppldpq eldilktvke itgflliqaw penrtdlhaf
    421 enleiirgrt kqhgqfslav vslnitslgl rslkeisdgd viisgnknlc yantinwkkl
    481 fgtsgqktki isnrgensck atgqvchalc spegcwgpep rdcvscrnvs rgrecvdkcn
    541 llegeprefv enseciqchp eclpqamnit ctgrgpdnci qcahyidgph cvktcpagvm
    601 genntivwky adaghvchlc hpnctygctg pglegcptng pkipsiatgm vgalllllvv
    661 algiglfmrr rhivrkrtlr rllqerelve pltpsgeapn qallrilket efkkikvlgs
    721 gafgtvykgl wipegekvki pvaikelrea tspkankeil deayvmasvd nphvcrllgi
    781 cltstvqlit qlmpfgclld yvrehkdnig sqyllnwcvq iakgmnyled rrlvhrdlaa
    841 rnvlvktpqh vkitdfglak llgaeekeyh aeggkvpikw malesilhri ythqsdvwsy
    901 gvtvwelmtf gskpydgipa seissilekg erlpqppict idvymimvkc wmidadsrpk
    961 freliiefsk mardpqrylv iqgdermhlp sptdsnfyra lmdeedmddv vdadeylipq
    1021 qgffsspsts rtpllsslsa tsnnstvaci drnglqscpi kedsflqrys sdptgalted
    1081 siddtflpvp gewlvwkqsc sstssthsaa aslqcpsqvl ppaspegetv adlqtq
    Epidermal growth factor receptor, isoform g precursor NP_001333828.1 (SEQ ID
    NO: 228)
    1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev
    61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala
    121 vlsnydankt glkelpmrnl qgqkcdpscp ngscwgagee ncqkltkiic aqqcsgrcrg
    181 kspsdcchnq caagctgpre sdclvcrkfr deatckdtcp plmlynptty qmdvnpegky
    241 sfgatcvkkc prnyvvtdhg scvracgads yemeedgvrk ckkcegperk vcngigigef
    301 kdslsinatn ikhfknctsi sgdlhilpva frgdsfthtp pldpqeldil ktvkeitgfl
    361 liqawpenrt dlhafenlei irgrtkqhgq fslavvslni tslglrslke isdgdviisg
    421 nknlcyanti nwkklfgtsg qktkiisnrg ensckatgqv chalcspegc wgpeprdcvs
    481 crnvsrgrec vdkcnllege prefvensec iqchpeclpq amnitctgrg pdnciqcahy
    541 idgphcvktc pagvmgennt lvwkyadagh vchlchpnct ygctgpgleg cptngpkips
    601 iatgmvgall lllvvalgig lfmrrrhivr krtlrrllqe relvepltps geapnqallr
    661 ilketefkki kvlgsgafgt vykglwipeg ekvkipvaik elreatspka nkeildeayv
    721 masvdnphvc rllgicltst vqlitqlmpf gclldyvreh kdnigsqyll nwcvqiakgm
    781 nyledrrlvh rdlaarnvlv ktpqhvkitd fglakllgae ekeyhaeggk vpikwmales
    841 ilhriythqs dvwsygvtvw elmtfgskpy dgipaseiss ilekgerlpq ppictidvym
    901 imvkcwmida dsrpkfreli iefskmardp qrylviqgde rmhlpsptds nfyralmdee
    961 dmddvvdade ylipqqgffs spstsrtpll sslsatsnns tvacidrngl qscpikedsf
    1021 lqryssdptg altedsiddt flpvpeyinq svpkrpagsv qnpvyhnqpl npapsrdphy
    1081 qdphstavgn peylntvqpt cvnstfdspa hwaqkgshqi sldnpdyqqd ffpkeakpng
    1141 ifkgstaena eylrvapqss efiga
    Epidermal growth factor receptor, isoform h NP_001333829.1 (SEQ ID NO: 229)
    1 mfnncevvlg nleityvqrn ydlsflktiq evagyvlial ntveriplen lqiirgnmyy
    61 ensyalavls nydanktglk elpmrnlqei lhgavrfsnn palcnvesiq wrdivssdfl
    121 snmsmdfqnh lgscqkcdps cpngscwgag eencqkltki icaqqcsgrc rgkspsdcch
    181 nqcaagctgp resdclvcrk frdeatckdt cpplmlynpt tyqmdvnpeg kysfgatcvk
    241 kcprnyvvtd hgscvracga dsyemeedgv rkckkcegpc rkvcngigig efkdslsina
    301 tnikhfknct sisgdlhilp vafrgdsfth tppldpqeld ilktvkeitg flliqawpen
    361 rtdlhafenl eiirgrtkqh gqfslavvsl nitslglrsl keisdgdvii sgnknlcyan
    421 tinwkklfgt sgqktkiisn rgensckatg qvchalcspe gcwgpeprdc vscrnvsrgr
    481 ecvdkcnlle geprefvens eciqchpecl pqamnitctg rgpdnciqca hyidgphcvk
    541 tcpagvmgen ntlvwkyada ghvchlchpn ctygctgpgl egcptngpki psiatgmvga
    601 lllllvvalg iglfmrrrhi vrkrtlrrll qerelveplt psgeapnqal lrilketefk
    661 kikvlgsgaf gtvykglwip egekvkipva ikelreatsp kankeildea yvmasvdnph
    721 vcrllgiclt stvqlitqlm pfgclldyvr ehkdnigsqy llnwcvqiak gmnyledrrl
    781 vhrdlaarnv lvktpqhvki tdfglakllg aeekeyhaeg gkvpikwmal esilhriyth
    841 qsdvwsygvt vwelmtfgsk pydgipasei ssilekgerl pqppictidv ymimvkcwmi
    901 dadsrpkfre liiefskmar dpqrylviqg dermhlpspt dsnfyralmd eedmddvvda
    961 deylipqqgf fsspstsrtp llsslsatsn nstvacidrn glqscpiked sflqryssdp
    1021 tgaltedsid dtflpvpeyi nqsvpkrpag svqnpvyhnq plnpapsrdp hyqdphstav
    1081 gnpeylntvq ptcvnstfds pahwaqkgsh qisldnpdyq qdffpkeakp ngifkgstae
    1141 naeylrvapq ssefiga
    Epidermal growth factor receptor, isoform i precursor NP_001333870.1 (SEQ ID
    NO: 230)
    1 mrpsgtagaa llallaalcp asraleekkg nyvvtdhgsc vracgadsye meedgvrkck
    61 kcegperkvc ngigigefkd slsinatnik hfknctsisg dlhilpvafr gdsfthtppl
    121 dpqeldilkt vkeitgflli qawpenrtdl hafenleiir grtkqhgqfs lavvslnits
    181 lglrslkeis dgdviisgnk nlcyantinw kklfgtsgqk tkiisnrgen sckatgqvch
    241 alcspegcwg peprdcvscr nvsrgrecvd kcnllegepr efvenseciq chpeclpqam
    301 nitctgrgpd nciqcahyid gphcvktcpa gvmgenntlv wkyadaghvc hlchpnctyg
    361 ctgpglegcp tngpkipsia tgmvgallll lvvalgiglf mrrrhivrkr tlrrllqere
    421 lvepltpsge apnqallril ketefkkikv lgsgafgtvy kglwipegek vkipvaikel
    481 reatspkank eildeayvma svdnphvcrl lgicltstvq litqlmpfgc lldyvrehkd
    541 nigsqyllnw cvqiakgmny ledrrlvhrd laarnvlvkt pqhvkitdfg lakllgaeek
    601 eyhaeggkvp ikwmalesil hriythqsdv wsygvtvwel mtfgskpydg ipaseissil
    661 ekgerlpqpp ictidvymim vkcwmidads rpkfreliie fskmardpqr ylviqgderm
    721 hlpsptdsnf yralmdeedm ddvvdadeyl ipqqgffssp stsrtpllss lsatsnnstv
    781 acidrnglqs cpikedsflq ryssdptgal tedsiddtfl pvpeyingsv pkrpagsvqn
    841 pvyhnqplnp apsrdphyqd phstavgnpe ylntvqptcv nstfdspahw aqkgshqisl
    901 dnpdyqqdff pkeakpngif kgstaenaey lrvapqssef iga
    Epithelial cell adhesion molecule NP_002345.2 (SEQ ID NO: 231)
    1 mappqvlafg lllaaatatf aaaqeecvce nyklavncfv nnnrqcqcts vgaqntvics
    61 klaakclvmk aemngsklgr rakpegalqn ndglydpdcd esglfkakqc ngtsmcwcvn
    121 tagvrrtdkd teitcservr tywiiielkh karekpydsk slrtalqkei ttryqldpkf
    181 itsilyennv itidlvqnss qktqndvdia dvayyfekdv kgeslfhskk mdltvngeql
    241 dldpgqtliy yvdekapefs mqglkagvia vivvvviavv agivvlvisr kkrmakyeka
    301 eikemgemhr elna
    Ephrin type-A receptor 2, isoform 1 precursor NP_004422.2 (SEQ ID NO: 232)
    1 melqaaracf allwgcalaa aaaaqgkevv lldfaaagge lgwlthpygk gwdlmqnimn
    61 dmpiymysvc nvmsgdqdnw lrtnwvyrge aerifielkf tvrdcnsfpg gasscketfn
    121 lyyaesdldy gtnfqkrlft kidtiapdei tvssdfearh vklnveersv gpltrkgfyl
    181 afqdigacva llsvrvyykk cpellqglah fpetiagsda pslatvagtc vdhavvppgg
    241 eeprmhcavd gewlvpigqc lcqagyekve dacqacspgf fkfeasespc lecpehtlps
    301 pegatscece egffrapqdp asmpctrpps aphyltavgm gakvelrwtp pqdsggredi
    361 vysvtceqcw pesgecgpce asvrysepph gltrtsvtvs dlephmnytf tvearngvsg
    421 lvtsrsfrta sysinqtepp kvrlegrstt slsvswsipp pqqsrvwkye vtyrkkgdsn
    481 synvrrtegf svtlddlapd ttylvqvgal tqegqgagsk vhefqtlspe gsgnlavigg
    541 vavgvvlllv lagvgffihr rrknqrarqs pedvyfskse qlkplktyvd phtyedpnqa
    601 vlkftteihp scvtrqkvig agefgevykg mlktssgkke vpvaiktlka gytekqrvdf
    661 lgeagimgqf shhniirleg viskykpmmi iteymengal dkflrekdge fsvlqlvgml
    721 rgiaagmkyl anmnyvhrdl aarnilvnsn lvckvsdfgl srvleddpea tyttsggkip
    781 irwtapeais yrkftsasdv wsfgivmwev mtygerpywe lsnhevmkai ndgfrlptpm
    841 dcpsaiyqlm mqcwqqerar rpkfadivsi ldklirapds lktladfdpr vsirlpstsg
    901 segvpfrtvs ewlesikmqq ytehfmaagy taiekvvqmt nddikrigvr lpghqkriay
    961 sllglkdqvn tvgipi
    Ephrin type-A receptor 2, isoform 2 NP_001316019.1 (SEQ ID NO: 233)
    1 mqnimndmpi ymysvcnvms gdqdnwlrtn wvyrgeaeri fielkftvrd cnsfpggass
    61 cketfnlyya esdldygtnf qkrlftkidt iapdeitvss dfearhvkln veersvgplt
    121 rkgfylafqd igacvallsv rvyykkcpel lqglahfpet iagsdapsla tvagtcvdha
    181 vvppggeepr mhcavdgewl vpigqclcqa gyekvedacq acspgffkfe asespclecp
    241 ehtlpspega tsceceegff rapqdpasmp ctrppsaphy ltavgmgakv elrwtppqds
    301 ggredivysv tceqcwpesg ecgpceasvr ysepphgltr tsvtvsdlep hmnytftvea
    361 rngvsglvts rsfrtasysi nqteppkvrl egrsttslsv swsipppqqs rvwkyevtyr
    421 kkgdsnsynv rrtegfsvtl ddlapdttyl vqvqaltqeg qgagskvhef qtlspegsgn
    481 laviggvavg vvlllvlagv gffihrrrkn qrarqspedv yfskseqlkp lktyvdphty
    541 edpnqavlkf tteihpscvt rqkvigagef gevykgmlkt ssgkkevpva iktlkagyte
    601 kqrvdflgea gimgqfshhn iirlegvisk ykpmmiitey mengaldkfl rekdgefsvl
    661 qlvgmlrgia agmkylanmn yvhrdlaarn ilvnsnlvck vsdfglsrvl eddpeatytt
    721 sggkipirwt apeaisyrkf tsasdvwsfg ivmwevmtyg erpywelsnh evmkaindgf
    781 rlptpmdcps aiyqlmmqcw qgerarrpkf adivsildkl irapdslktl adfdprvsir
    841 lpstsgsegv pfrtvsewle sikmqqyteh fmaagytaie kvvqmtnddi krigvrlpgh
    901 qkriaysllg lkdqvntvgi pi
    Receptor-tyrosine-protein kinase erbB-2, isoform a precursor NP_004439.2 (SEQ
    ID NO: 234)
    1 melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly qgcqvvqgnl
    61 eltylptnas lsflqdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng
    121 dpinnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdtilwk difhknnqla
    181 ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc
    241 aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp
    301 ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan
    361 iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf etleeitgyl yisawpdslp
    421 dlsvfqnlqv irgrilhnga ysltlqglgi swlglrslre lgsglalihh nthlcfvhtv
    481 pwdqlfrnph qallhtanrp edecvgegla chqlcarghc wgpgptqcvn csqflrgqec
    541 veecrvlqgl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc
    601 psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisavvg
    661 illvvvlgvv fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel
    721 rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp
    781 yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr
    841 lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft
    901 hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm
    961 idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl dstfyrslle dddmgdlvda
    1021 eeylvpqqgf fcpdpapgag gmvhhrhrss strsgggdlt lglepseeea prsplapseg
    1081 agsdvfdgdl gmgaakglqs lpthdpsplq rysedptvpl psetdgyvap ltcspqpeyv
    1141 nqpdvrpqpp spregplpaa rpagatlerp ktlspgkngv vkdvfafgga venpeyltpq
    1201 ggaapqphpp pafspafdnl yywdqdpper gappstfkgt ptaenpeylg ldvpv
    Receptor-tyrosine-protein kinase erbB-2, isoform b NP_001005862.1 (SEQ ID NO:
    235)
    1 mklrlpaspe thldmlrhly qgcqvvqgnl eltylptnas lsflqdiqev qgyvliahnq
    61 vrqvplqrlr ivrgtqlfed nyalavldng dpinnttpvt gaspgglrel qlrslteilk
    121 ggvliqrnpq lcyqdtilwk difhknnqla ltlidtnrsr achpcspmck gsrcwgesse
    181 dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs dclaclhfnh sgicelhcpa
    241 lvtyntdtfe smpnpegryt fgascvtacp ynylstdvgs ctlvcplhnq evtaedgtqr
    301 cekcskpcar vcyglgmehl revravtsan iqefagckki fgslaflpes fdgdpasnta
    361 plqpeqlqvf etleeitgyl yisawpdslp dlsvfqnlqv irgrilhnga ysltlqglgi
    421 swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qallhtanrp edecvgegla
    481 chqlcarghc wgpgptqcvn csqflrgqec veecrvlqgl preyvnarhc lpchpecqpq
    541 ngsvtcfgpe adqcvacahy kdppfcvarc psgvkpdlsy mpiwkfpdee gacqpcpinc
    601 thscvdlddk gcpaeqrasp ltsiisavvg illvvvlgvv fgilikrrqq kirkytmrrl
    661 lqetelvepl tpsgampnqa qmrilketel rkvkvlgsga fgtvykgiwi pdgenvkipv
    721 aikvlrents pkankeilde ayvmagvgsp yvsrllgicl tstvqlvtql mpygclldhv
    781 renrgrlgsq dllnwcmqia kgmsyledvr lvhrdlaarn vlvkspnhvk itdfglarll
    841 dideteyhad ggkvpikwma lesilrrrft hqsdvwsygv tvwelmtfga kpydgipare
    901 ipdllekger lpqppictid vymimvkcwm idsecrprfr elvsefsrma rdpqrfvviq
    961 nedlgpaspl dstfyrslle dddmgdlvda eeylvpqqgf fcpdpapgag gmvhhrhrss
    1021 strsgggdlt lglepseeea prsplapseg agsdvfdgdl gmgaakglqs lpthdpsplq
    1081 rysedptvpl psetdgyvap ltcspqpeyv nqpdvrpqpp spregplpaa rpagatlerp
    1141 ktlspgkngv vkdvfafgga venpeyltpq ggaapqphpp pafspafdnl yywdqdpper
    1201 gappstfkgt ptaenpeylg ldvpv
    Receptor-tyrosine-protein kinase erbB-2, isoform c NP_001276865.1 (SEQ ID NO:
    236)
    1 mprgswkpqv ctgtdmklrl paspethldm lrhlyqgcqv vqgnleltyl ptnaslsflq
    61 diqevqgyvl iahnqvrqvp lqrlrivrgt qlfednyala vldngdpinn ttpvtgaspg
    121 glrelqlrsl teilkggvli grnpqlcyqd tilwkdifhk nnqlaltlid tnrsrachpc
    181 spmckgsrcw gessedcqsl trtvcaggca rckgplptdc cheqcaagct gpkhsdclac
    241 lhfnhsgice lhcpalvtyn tdtfesmpnp egrytfgasc vtacpynyls tdvgsctlvc
    301 plhnqevtae dgtqrcekcs kpcarvcygl gmehlrevra vtsaniqefa gckkifgsla
    361 flpesfdgdp asntaplqpe qlqvfetlee itgylyisaw pdslpdlsvf qnlqvirgri
    421 lhngaysltl qglgiswlgl rslrelgsgl alihhnthlc fvhtvpwdql frnphqallh
    481 tanrpedecv geglachqlc arghcwgpgp tqcvncsqfl rgqecveecr vlqglpreyv
    541 narhclpchp ecqpqngsvt cfgpeadqcv acahykdppf cvarcpsgvk pdlsympiwk
    601 fpdeegacqp cpincthscv dlddkgcpae qraspltsii savvgillvv vlgvvfgili
    661 krrqqkirky tmrrllqete lvepltpsga mpnqaqmril ketelrkvkv lgsgafgtvy
    721 kgiwipdgen vkipvaikvl rentspkank eildeayvma gvgspyvsrl lgicltstvq
    781 lvtqlmpygc lldhvrenrg rlgsqdllnw cmqiakgmsy ledvrlvhrd laarnvlvks
    841 pnhvkitdfg larlldidet eyhadggkvp ikwmalesil rrrfthqsdv wsygvtvwel
    901 mtfgakpydg ipareipdll ekgerlpqpp ictidvymim vkcwmidsec rprfrelvse
    961 fsrmardpqr fvviqnedlg paspldstfy rslledddmg dlvdaeeylv pqqgffcpdp
    1021 apgaggmvhh rhrssstrsg ggdltlglep seeeaprspl apsegagsdv fdgdlgmgaa
    1081 kglqslpthd psplqrysed ptvplpsetd gyvapltcsp qpeyvnqpdv rpqppspreg
    1141 plpaarpaga tlerpktlsp gkngvvkdvf afggavenpe yltpqggaap qphpppafsp
    1201 afdnlyywdq dppergapps tfkgtptaen peylgldvpv
    Receptor-tyrosine-protein kinase erbB-2, isoform d NP_001276866.1 (SEQ ID NO:
    237)
    1 melaalcrwg lllallppga astqvctgtd mklrlpaspe thldmlrhly qgcqvvqgnl
    61 eltylptnas lsflqdiqev qgyvliahnq vrqvplqrlr ivrgtqlfed nyalavldng
    121 dpinnttpvt gaspgglrel qlrslteilk ggvliqrnpq lcyqdtilwk difhknnqla
    181 ltlidtnrsr achpcspmck gsrcwgesse dcqsltrtvc aggcarckgp lptdccheqc
    241 aagctgpkhs dclaclhfnh sgicelhcpa lvtyntdtfe smpnpegryt fgascvtacp
    301 ynylstdvgs ctlvcplhnq evtaedgtqr cekcskpcar vcyglgmehl revravtsan
    361 iqefagckki fgslaflpes fdgdpasnta plqpeqlqvf etleeitgyl yisawpdslp
    421 dlsvfqnlqv irgrilhnga ysltlqglgi swlglrslre lgsglalihh nthlcfvhtv
    481 pwdqlfrnph qallhtanrp edecvgegla chqlcarghc wgpgptqcvn csqflrgqec
    541 veecrvlqgl preyvnarhc lpchpecqpq ngsvtcfgpe adqcvacahy kdppfcvarc
    601 psgvkpdlsy mpiwkfpdee gacqpcpinc thscvdlddk gcpaeqrasp ltsiisavvg
    661 illvvvlgvv fgilikrrqq kirkytmrrl lqetelvepl tpsgampnqa qmrilketel
    721 rkvkvlgsga fgtvykgiwi pdgenvkipv aikvlrents pkankeilde ayvmagvgsp
    781 yvsrllgicl tstvqlvtql mpygclldhv renrgrlgsq dllnwcmqia kgmsyledvr
    841 lvhrdlaarn vlvkspnhvk itdfglarll dideteyhad ggkvpikwma lesilrrrft
    901 hqsdvwsygv tvwelmtfga kpydgipare ipdllekger lpqppictid vymimvkcwm
    961 idsecrprfr elvsefsrma rdpqrfvviq nedlgpaspl dstfyrslle dddmgdlvda
    1021 eeylvpqqgf fcpdpapgag gmvhhrhrss strnm
    Receptor-tyrosine-protein kinase erbB-2, isoform e NP_001276867.1 (SEQ ID NO:
    238)
    1 mklrlpaspe thldmlrhly qgcqvvqgnl eltylptnas lsflqdiqev qgyvliahnq
    61 vrqvplqrlr ivrgtqlfed nyalavldng dpinnttpvt gaspgglrel qlrslteilk
    121 ggvliqrnpq lcyqdtilwk difhknnqla ltlidtnrsr achpcspmck gsrcwgesse
    181 dcqsltrtvc aggcarckgp lptdccheqc aagctgpkhs dclaclhfnh sgicelhcpa
    241 lvtyntdtfe smpnpegryt fgascvtacp ynylstdvgs ctlvcplhnq evtaedgtqr
    301 cekcskpcar vcyglgmehl revravtsan iqefagckki fgslaflpes fdgdpasnta
    361 plqpeqlqvf etleeitgyl yisawpdslp dlsvfqnlqv irgrilhnga ysltlqglgi
    421 swlglrslre lgsglalihh nthlcfvhtv pwdqlfrnph qallhtanrp edecvgegla
    481 chqlcarghc wgpgptqcvn csqflrgqec veecrvlqgl preyvnarhc lpchpecqpq
    541 ngsvtcfgpe adqcvacahy kdppfcvarc psgvkpdlsy mpiwkfpdee gacqpcpinc
    601 ths
    Receptor tyrosine-protein kinase erbB-4, isoform JM-a/CVT-1 precursor
    NP_005226.1 (SEQ ID NO: 239)
    1 mkpatglwvw vsllvaagtv qpsdsqsvca gtenklssls dleqqyralr kyyencevvm
    61 gnleitsieh nrdlsflrsv revtgyvlva lnqfrylple nlriirgtkl yedryalaif
    121 lnyrkdgnfg lqelglknit eilnggvyvd qnkflcyadt ihwqdivrnp wpsnltivst
    181 ngssgcgrch ksctgrcwgp tenhcqtltr tvcaeqcdgr cygpyvsdcc hrecaggcsg
    241 pkdtdcfacm nfndsgacvt qcpqtfvynp ttfqlehnfn akytygafcv kkcphnfvvd
    301 ssscvracps skmeveengi kmckpctdic pkacdgigtg slmsaqtvds snidkfinct
    361 kingnliflv tgihgdpyna ieaidpekln vfrtvreitg flniqswppn mtdfsvfsnl
    421 vtiggrvlys glsllilkqq gitslqfqsl keisagniyi tdnsnlcyyh tinwttlfst
    481 inqrivirdn rkaenctaeg mvcnhlcssd gcwgpgpdqc lscrrfsrgr iciescnlyd
    541 gefrefengs icvecdpqce kmedglltch gpgpdnctkc shfkdgpncv ekcpdglqga
    601 nsfifkyadp drechpchpn ctqgcngpts hdciyypwtg hstlpqhart pliaagvigg
    661 lfilvivglt favyvrrksi kkkralrrfl etelvepltp sgtapnqaql rilketelkr
    721 vkvlgsgafg tvykgiwvpe getvkipvai kilnettgpk anvefmdeal imasmdhphl
    781 vrllgvclsp tiqlvtqlmp hgclleyvhe hkdnigsqll lnwcvqiakg mmyleerrlv
    841 hrdlaarnvl vkspnhvkit dfglarlleg dekeynadgg kmpikwmale cihyrkfthq
    901 sdvwsygvti welmtfggkp ydgiptreip dllekgerlp qppictidvy mvmvkcwmid
    961 adsrpkfkel aaefsrmard pqrylviqgd drmklpspnd skffqnllde edledmmdae
    1021 eylvpqafni pppiytsrar idsnrseigh spppaytpms gnqfvyrdgg faaeqgvsvp
    1081 yraptstipe apvaqgatae ifddsccngt lrkpvaphvq edsstqrysa dptvfapers
    1141 prgeldeegy mtpmrdkpkq eylnpveenp fvsrrkngdl qaldnpeyhn asngppkaed
    1201 eyvneplyln tfantlgkae ylknnilsmp ekakkafdnp dywnhslppr stlqhpdylq
    1261 eystkyfykq ngrirpivae npeylsefsl kpgtvlpppp yrhrntvv
    Receptor tyrosine-protein kinase erbB-4, isoform JM-a/CVT-2 precursor
    NP_001036064.1 (SEQ ID NO: 240)
    1 mkpatglwvw vsllvaagtv qpsdsqsvca gtenklssls dleqqyralr kyyencevvm
    61 gnleitsieh nrdlsflrsv revtgyvlva lnqfrylple nlriirgtkl yedryalaif
    121 lnyrkdgnfg lqelglknit eilnggvyvd qnkflcyadt ihwqdivrnp wpsnltlvst
    181 ngssgcgrch ksctgrcwgp tenhcqtltr tvcaeqcdgr cygpyvsdcc hrecaggcsg
    241 pkdtdcfacm nfndsgacvt qcpqtfvynp ttfqlehnfn akytygafcv kkcphnfvvd
    301 ssscvracps skmeveengi kmckpctdic pkacdgigtg slmsaqtvds snidkfinct
    361 kingnliflv tgihgdpyna ieaidpekln vfrtvreitg flniqswppn mtdfsvfsnl
    421 vtiggrvlys glsllilkqq gitslqfqsl keisagniyi tdnsnlcyyh tinwttlfst
    481 inqrivirdn rkaenctaeg mvcnhlcssd gcwgpgpdqc lscrrfsrgr iciescnlyd
    541 gefrefengs icvecdpqce kmedglltch gpgpdnctkc shfkdgpncv ekcpdglqga
    601 nsfifkyadp drechpchpn ctqgcngpts hdciyypwtg hstlpqhart pliaagvigg
    661 lfilvivglt favyvrrksi kkkralrrfl etelvepltp sgtapnqaql rilketelkr
    721 vkvlgsgafg tvykgiwvpe getvkipvai kilnettgpk anvefmdeal imasmdhphl
    781 vrllgvclsp tiqlvtqlmp hgclleyvhe hkdnigsqll lnwcvqiakg mmyleerrlv
    841 hrdlaarnvl vkspnhvkit dfglarlleg dekeynadgg kmpikwmale cihyrkfthq
    901 sdvwsygvti welmtfggkp ydgiptreip dllekgerlp qppictidvy mvmvkcwmid
    961 adsrpkfkel aaefsrmard pqrylviqgd drmklpspnd skffqnllde edledmmdae
    1021 eylvpqafni pppiytsrar idsnrnqfvy rdggfaaeqg vsvpyrapts tipeapvaqg
    1081 ataeifddsc cngtlrkpva phvqedsstq rysadptvfa persprgeld eegymtpmrd
    1141 kpkqeylnpv eenpfvsrrk ngdlqaldnp eyhnasngpp kaedeyvnep lylntfantl
    1201 gkaeylknni lsmpekakka fdnpdywnhs lpprstlqhp dylqeystky fykqngrirp
    1261 ivaenpeyls efslkpgtvl ppppyrhrnt vv
    Prolyl endopeptidase FAP, isoform 1 NP_004451.2 (SEQ ID NO: 241)
    1 mktwvkivfg vatsavlall vmcivlrpsr vhnseentmr altlkdilng tfsyktffpn
    61 wisgqeylhq sadnnivlyn ietgqsytil snrtmksvna snyglspdrq fvylesdysk
    121 lwrysytaty yiydlsngef vrgnelprpi qylcwspvgs klayvyqnni ylkqrpgdpp
    181 fqitfngren kifngipdwv yeeemlatky alwwspngkf layaefndtd ipviaysyyg
    241 deqyprtini pypkagaknp vvrifiidtt ypayvgpqev pvpamiassd yyfswltwvt
    301 dervclqwlk rvqnvsvlsi cdfredwqtw dcpktqehie esrtgwaggf fvstpvfsyd
    361 aisyykifsd kdgykhihyi kdtvenaiqi tsgkweaini frvtqdslfy ssnefeeypg
    421 rrniyrisig syppskkcvt chlrkercqy ytasfsdyak yyalvcygpg ipistlhdgr
    481 tdqeikilee nkelenalkn iqlpkeeikk levdeitlwy kmilppqfdr skkyplliqv
    541 yggpcsqsvr svfavnwisy laskegmvia lvdgrgtafq gdkllyavyr klgvyevedq
    601 itavrkfiem gfidekriai wgwsyggyvs slalasgtgl fkcgiavapv ssweyyasvy
    661 terfmglptk ddnlehykns tvmaraeyfr nvdyllihgt addnvhfqns aqiakalvna
    721 qvdfqamwys dqnhglsgls tnhlythmth flkqcfslsd
    Prolyl endopeptidase FAP, isoform 2 NP_001278736.1 (SEQ ID NO: 242)
    1 mktwvkivfg vatsavlall vmcivlrpsr vhnseentmr altlkdilng tfsyktffpn
    61 wisgqeylhq sadnnivlyn ietgqsytil snrtmlwrys ytatyyiydl sngefvrgne
    121 lprpiqylcw spvgsklayv yqnniylkqr pgdppfqitf ngrenkifng ipdwvyeeem
    181 latkyalwws pngkflayae fndtdipvia ysyygdeqyp rtinipypka gaknpvvrif
    241 iidttypayv gpqevpvpam iassdyyfsw ltwvtdervc lqwlkrvqnv svlsicdfre
    301 dwqtwdcpkt qehieesrtg waggffvstp vfsydaisyy kifsdkdgyk hihyikdtve
    361 naiqitsgkw eainifrvtq dslfyssnef eeypgrrniy risigsypps kkcvtchlrk
    421 ercqyytasf sdyakyyalv cygpgipist lhdgrtdqei kileenkele nalkniqlpk
    481 eeikklevde itlwykmilp pqfdrskkyp lliqvyggpc sqsvrsvfav nwisylaske
    541 gmvialvdgr gtafqgdkll yavyrklgvy evedqitavr kfiemgfide kriaiwgwsy
    601 ggyvsslala sgtglfkcgi avapvsswey yasvyterfm glptkddnle hyknstvmar
    661 aeyfrnvdyl lihgtaddnv hfqnsaqiak alvnaqvdfq amwysdqnhg lsglstnhly
    721 thmthflkqc fslsd
    Glutamate carboxypeptidase 2, isoform 1 NP_004467.1 (SEQ ID NO: 243)
    1 mwnllhetds avatarrprw lcagalvlag gffllgflfg wfikssneat nitpkhnmka
    61 fldelkaeni kkflynftqi phlagteqnf qlakqiqsqw kefgldsvel ahydvllsyp
    121 nkthpnyisi inedgneifn tslfeppppg yenvsdivpp fsafspqgmp egdlvyvnya
    181 rtedffkler dmkincsgki viarygkvfr gnkvknaqla gakgvilysd padyfapgvk
    241 sypdgwnlpg ggvqrgniln lngagdpltp gypaneyayr rgiaeavglp sipvhpigyy
    301 daqkllekmg gsappdsswr gslkvpynvg pgftgnfstq kvkmhihstn evtriynvig
    361 tlrgavepdr yvilgghrds wvfggidpqs gaavvheivr sfgtlkkegw rprrtilfas
    421 wdaeefgllg stewaeensr llqergvayi nadssiegny tlrvdctplm yslvhnitke
    481 lkspdegfeg kslyeswtkk spspefsgmp risklgsgnd fevffqrlgi asgrarytkn
    541 wetnkfsgyp lyhsvyetye lvekfydpmf kyhltvaqvr ggmvfelans ivlpfdcrdy
    601 avvlrkyadk iysismkhpq emktysysfd slfsavknft eiaskfserl qdfdksnpiv
    661 lrmmndqlmf lerafidplg lpdrpfyrhv iyapsshnky agesfpgiyd alfdieskvd
    721 pskawgevkr qiyvaaftvq aaaetlseva
    Glutamate carboxypeptidase 2, isoform 2 NP_001014986.1 (SEQ ID NO: 244)
    1 mwnllhetds avatarrprw lcagalvlag gffllgflfg wfikssneat nitpkhnmka
    61 fldelkaeni kkflynftqi phlagteqnf qlakqiqsqw kefgldsvel ahydvllsyp
    121 nkthpnyisi inedgneifn tslfeppppg yenvsdivpp fsafspqgmp egdlvyvnya
    181 rtedffkler dmkincsgki viarygkvfr gnkvknaqla gakgvilysd padyfapgvk
    241 sypdgwnlpg ggvqrgniln lngagdpltp gypaneyayr rgiaeavglp sipvhpigyy
    301 daqkllekmg gsappdsswr gslkvpynvg pgftgnfstq kvkmhihstn evtriynvig
    361 tlrgavepdr yvilgghrds wvfggidpqs gaavvheivr sfgtlkkegw rprrtilfas
    421 wdaeefgllg stewaeensr llqergvayi nadssiegny tlrvdctplm yslvhnitke
    481 lkspdegfeg kslyeswtkk spspefsgmp risklgsgnd fevffqrlgi asgrarytkn
    541 wetnkfsgyp lyhsvyetye lvekfydpmf kyhltvaqvr ggmvfelans ivlpfdcrdy
    601 avvlrkyadk iysismkhpq emktysysfd slfsavknft eiaskfserl qdfdkskhvi
    661 yapsshnkya gesfpgiyda lfdieskvdp skawgevkrq iyvaaftvqa aaetlseva
    Glutamate carboxypeptidase 2, isoform 3 NP_001180400.1 (SEQ ID NO: 245)
    1 mtagssyplf laayactgcl aerlgwfiks sneatnitpk hnmkafldel kaenikkfly
    61 nftqiphlag teqnfqlakq iqsqwkefgl dsvelahydv llsypnkthp nyisiinedg
    121 neifntslfe ppppgyenvs divppfsafs pqgmpegdlv yvnyartedf fklerdmkin
    181 csgkiviary gkvfrgnkvk naqlagakgv ilysdpadyf apgvksypdg wnlpgggvqr
    241 gnilnlngag dpltpgypan eyayrrgiae avglpsipvh pigyydaqkl lekmggsapp
    301 dsswrgslkv pynvgpgftg nfstqkvkmh ihstnevtri ynvigtlrga vepdryvilg
    361 ghrdswvfgg idpqsgaavv heivrsfgtl kkegwrprrt ilfaswdaee fgllgstewa
    421 eensrllqer gvayinadss iegnytlrvd ctplmyslvh nitkelkspd egfegkslye
    481 swtkkspspe fsgmpriskl gsgndfevff qrlgiasgra rytknwetnk fsgyplyhsv
    541 yetyelvekf ydpmfkyhlt vaqvrggmvf elansivlpf dcrdyavvlr kyadkiysis
    601 mkhpqemkty sysfdslfsa vknfteiask fserlqdfdk snpivlrmmn dqlmfleraf
    661 idplglpdrp fyrhviyaps shnkyagesf pgiydalfdi eskvdpskaw gevkrqiyva
    721 aftvqaaaet lseva
    Glutamate carboxypeptidase 2, isoform 4 NP_001180401.1 (SEQ ID NO: 246)
    1 mtagssyplf laayactgcl aerlgwfiks sneatnitpk hnmkafldel kaenikkfly
    61 nftqiphlag teqnfqlakq iqsqwkefgl dsvelahydv llsypnkthp nyisiinedg
    121 neifntslfe ppppgyenvs divppfsafs pqgmpegdlv yvnyartedf fklerdmkin
    181 csgkiviary gkvfrgnkvk naqlagakgv ilysdpadyf apgvksypdg wnlpgggvqr
    241 gnilnlngag dpltpgypan eyayrrgiae avglpsipvh pigyydaqkl lekmggsapp
    301 dsswrgslkv pynvgpgftg nfstqkvkmh ihstnevtri ynvigtlrga vepdryvilg
    361 ghrdswvfgg idpqsgaavv heivrsfgtl kkegwrprrt ilfaswdaee fgllgstewa
    421 eensrllqer gvayinadss iegnytlrvd ctplmyslvh nitkelkspd egfegkslye
    481 swtkkspspe fsgmpriskl gsgndfevff qrlgiasgra rytknwetnk fsgyplyhsv
    541 yetyelvekf ydpmfkyhlt vaqvrggmvf elansivlpf dcrdyavvlr kyadkiysis
    601 mkhpqemkty sysfdslfsa vknfteiask fserlqdfdk skhviyapss hnkyagesfp
    661 giydalfdie skvdpskawg evkrqiyvaa ftvqaaaetl seva
    Glutamate carboxypeptidase 2, isoform 5 NP_001180402.1 (SEQ ID NO: 247)
    1 mggsappdss wrgslkvpyn vgpgftgnfs tqkvkmhihs tnevtriynv igtlrgavep
    61 dryvilgghr dswvfggidp qsgaavvhei vrsfgtlkke gwrprrtilf aswdaeefgl
    121 lgstewaeen srllqergva yinadssieg nytlrvdctp lmyslvhnit kelkspdegf
    181 egkslyeswt kkspspefsg mprisklgsg ndfevffqrl giasgraryt knwetnkfsg
    241 yplyhsvyet yelvekfydp mfkyhltvaq vrggmvfela nsivlpfdcr dyavvlrkya
    301 dkiysismkh pqemktysvs fdslfsavkn fteiaskfse rlqdfdksnp ivlrmmndql
    361 mflerafidp lglpdrpfyr hviyapsshn kyagesfpgi ydalfdiesk vdpskawgev
    421 krqiyvaaft vqaaaetlse va
    Glutamate carboxypeptidase 2, isoform 6 NP_001338165.1 (SEQ ID NO: 248)
    1 mkafldelka enikkflynf tqiphlagte qnfqlakqiq sqwkefglds velahydvll
    61 sypnkthpny isiinedgne ifntslfepp ppgyenvsdi vppfsafspq gmpegdlvyv
    121 nyartedffk lerdmkincs gkiviarygk vfrgnkvkna qlagakgvil ysdpadyfap
    181 gvksypdgwn lpgggvqrgn ilnlngagdp ltpgypaney ayrrgiaeav glpsipvhpi
    241 gyydaqklle kmggsappds swrgslkvpy nvgpgftgnf stqkvkmhih stnevtriyn
    301 vigtlrgave pdryvilggh rdswvfggid pqsgaavvhe ivrsfgtlkk egwrprrtil
    361 faswdaeefg llgstewaee nsrllqergv ayinadssie gnytlrvdct plmyslvhnl
    421 tkelkspdeg fegkslyesw tkkspspefs gmprisklgs gndfevffqr lgiasgrary
    481 tknwetnkfs gyplyhsvye tyelvekfyd pmfkyhltva qvrggmvfel ansivlpfdc
    541 rdyavvlrky adkiysismk hpqemktysv sfdslfsavk nfteiaskfs erlqdfdksk
    601 hviyapsshn kyagesfpgi ydalfdiesk vdpskawgev krqiyvaaft vqaaaetlse
    661 va
    Fos-related antigen 1, isoform 1 NP_005429.1 (SEQ ID NO: 249)
    1 mfrdfgepgp ssgngggygg paqppaaaqa aqqkfhlvps intmsgsqel qwmvqphflg
    61 pssyprplty pqysppqprp gviralgppp gvrrrpceqi speeeerrrv rrernklaaa
    121 kcrnrrkelt dflqaetdkl edeksglqre ieelqkqker lelvleahrp ickipegake
    181 gdtgstsgts sppapcrpvp cislspgpvl epealhtptl mttpsltpft pslvftypst
    241 pepcasahrk sssssgdpss dplgsptlla l
    Fos-related antigen 1, isoform 2 NP_001287773.1 (SEQ ID NO: 250)
    1 mfrdfgepgp ssgngggygg paqppaaaqa aqqkfhlvps intmsgsqel qwmvqphflg
    61 pssyprplty pqysppqprp gviralgppp gvrrrpceqe tdkledeksg lqreieelqk
    121 qkerlelvle ahrpickipe gakegdtgst sgtssppapc rpvpcislsp gpvlepealh
    181 tptlmttpsl tpftpslvft ypstpepcas ahrksssssg dpssdplgsp tllal
    Fos-related antigen 1, isoform 3 NP_001287784.1 (SEQ ID NO: 251)
    1 mfrdfgepgp ssgngggygg paqppaaaqa aqqkfhlvps intmsgsqel qwmvqphflg
    61 pssyprplty pqysppqprp gviralgppp gvrrrpceqp ggrgappska raeqagcgqv
    121 gepeegtdrl paggd
    Fos-related antigen 1, isoform 4 NP_001287785.1 (SEQ ID NO: 252)
    1 mfrdfgepgp ssgngggygg paqppaaaqa aqqispeeee rrrvrrernk laaakcrnrr
    61 keltdflqae tdkledeksg lqreieelqk qkerlelvle ahrpickipe gakegdtgst
    121 sgtssppapc rpvpcislsp gpvlepealh tptlmttpsl tpftpslvft ypstpepcas
    181 ahrksssssg dpssdplgsp tllal
    Fos-related antigen 1, isoform 5 NP_001287786.1 (SEQ ID NO: 253)
    1 mfrdfgepgp ssgngggygg paqppaaaqa aqqetdkled eksglqreie elqkqkerle
    61 lvleahrpic kipegakegd tgstsgtssp paperpvpci slspgpvlep ealhtptlmt
    121 tpsltpftps lvftypstpe pcasahrkss sssgdpssdp lgsptllal
    G antigen
     1 NP_001035753.1 (SEQ ID NO: 254)
    1 mswrgrstyy wprprryvqp pemigpmrpe qfsdevepat peegepatqr qdpaaaqege
    61 degasagqgp kpeadsqeqg hpqtgceced gpdgqemdpp npeevktpee gegqsqc
    G antigen 12I NP_001465.1 (SEQ ID NO: 255)
    1 mswrgrstyy wprprryvqp pemigpmrpe qfsdevepat peegepatqr qdpaaaqege
    61 degasagqgp kpeadsqeqg hpqtgceced gpdgqemdpp npeevktpee gekqsqc
    Galectin-1 NP_002296.1 (SEQ ID NO: 256)
    1 macglvasnl nlkpgeclrv rgevapdaks fvinlgkdsn nlclhfnprf nahgdantiv
    61 cnskdggawg teqreavfpf qpgsvaevci tfdqanltvk lpdgyefkfp nrlnleainy
    121 maadgdfkik cvafd
    Galectin-3 isoform 1 NP_002297.2 (SEQ ID NO: 257)
    1 madnfslhda lsgsgnpnpq gwpgawgnqp agaggypgas ypgaypgqap pgaypgqapp
    61 gaypgapgay pgapapgvyp gppsgpgayp ssgqpsatga ypatgpygap agplivpynl
    121 plpggvvprm litilgtvkp nanrialdfq rgndvafhfn prfnennrrv ivcntkldnn
    181 wgreerqsvf pfesgkpfki qvlvepdhfk vavndahllq ynhrvkklne isklgisgdi
    241 dltsasytmi
    Galectin-3, isoform 3 NP_001344607.1 (SEQ ID NO: 258)
    1 mhsktpcgcf kpwkmadnfs lhdalsgsgn pnpqgwpgaw gnqpagaggy pgasypgayp
    61 gqappgaypg qappgaypga pgaypgapap gvypgppsgp gaypssgqps atgaypatgp
    121 ygapagpliv pynlplpggv vprmlitilg tvkpnanria ldfqrgndva fhfnprfnen
    181 nrrvivcntk ldnnwgreer qsvfpfesgk pfkiqvlvep dhfkvavnda hllqynhrvk
    241 klneisklgi sgdidltsas ytmi
    Galectin-9 short NP_002299.2 (SEQ ID NO: 259)
    1 mafsgsqapy lspavpfsgt iqgglqdglq itvngtvlss sgtrfavnfq tgfsgndiaf
    61 hfnprfedgg yvvcntrqng swgpeerkth mpfqkgmpfd lcflvqssdf kvmvngilfv
    121 qyfhrvpfhr vdtisvngsv qlsyisfqpp gvwpanpapi tqtvihtvqs apgqmfstpa
    181 ippmmyphpa ypmpfittil gglypsksil lsgtvlpsaq rfhinlcsgn hiafhlnprf
    241 denavvrntq idnswgseer slprkmpfvr gqsfsvwilc eahclkvavd gqhlfeyyhr
    301 lrnlptinrl evggdiqlth vqt
    Galectin-9 long NP_033665.1 (SEQ ID NO: 260)
    1 mafsgsqapy lspavpfsgt iqgglqdglq itvngtvlss sgtrfavnfq tgfsgndiaf
    61 hfnprfedgg yvvcntrqng swgpeerkth mpfqkgmpfd lcflvqssdf kvmvngilfv
    121 qyfhrvpfhr vdtisvngsv qlsyisfqnp rtvpvqpafs tvpfsqpvcf pprprgrrqk
    181 ppgvwpanpa pitqtvihtv qsapgqmfst paippmmyph paypmpfitt ilgglypsks
    241 illsgtvlps aqrfhinlcs gnhiafhlnp rfdenavvrn tqidnswgse erslprkmpf
    301 vrgqsfsvwi lceahclkva vdgqhlfeyy hrlrnlptin rlevggdiql thvqt
    Galectin-9 isoform 3 NP_001317092.1 (SEQ ID NO: 261)
    1 mafsgsqapy lspavpfsgt iqgglqdglq itvngtvlss sgtrfavnfq tgfsgndiaf
    61 hfnprfedgg yvvcntrqng swgpeerkth mpfqkgmpfd lcflvqssdf kvmvngilfv
    121 qyfhrvpfhr vdtisvngsv qlsyisfqpp gvwpanpapi tqtvihtvqs apgqmfstpa
    181 ippmmyphpa ypmpfittil gglypsksil lsgtvlpsaq rcgscvklta srwpwmvstc
    241 lnttia
    Premelanosome protein, isoform 1 preprotein NP_001186983.1 (SEQ ID NO: 262)
    1 mdlvlkrcll hlavigalla vgatkvprnq dwlgvsrqlr tkawnrqlyp ewteaqrldc
    61 wrggqvslkv sndgptliga nasfsialnf pgsqkvlpdg qviwvnntii ngsqvwggqp
    121 vypqetddac ifpdggpcps gswsqkrsfv yvwktwgqyw qvlggpvsgl sigtgramlg
    181 thtmevtvyh rrgsrsyvpl ahsssaftit dqvpfsysvs qlraldggnk hflrnqpltf
    241 alqlhdpsgy laeadlsytw dfgdssgtli sralvvthty lepgpvtaqv vlqaaiplts
    301 cgsspvpgtt dghrptaeap nttagqvptt evvgttpgqa ptaepsgtts vqvpttevis
    361 tapvqmptae stgmtpekvp vsevmgttla emstpeatgm tpaevsivvl sgttaaqvtt
    421 tewvettare lpipepegpd assimstesi tgslgplldg tatlrlvkrq vpldcvlyry
    481 gsfsvtldiv qgiesaeilq avpsgegdaf eltvscqggl pkeacmeiss pgcqppaqrl
    541 cqpvlpspac qlvlhqilkg gsgtyclnvs ladtnslavv stqlimpvpg illtgqeagl
    601 gqvplivgil lvlmavvlas liyrrrlmkq dfsvpqlphs sshwlrlpri fcscpigens
    661 pllsgqqv
    Premelanosome protein, isoform 2 precursor NP_001186982.1 (SEQ ID NO: 263)
    1 mdlvlkrcll hlavigalla vgatkgsqvw ggqpvypqet ddacifpdgg pcpsgswsqk
    61 rsfvyvwktw gqywqvlggp vsglsigtgr amlgthtmev tvyhrrgsrs yvplahsssa
    121 ftitdqvpfs vsysqlrald ggnkhflrnq pltfalqlhd psgylaeadl sytwdfgdss
    181 gtlisralvv thtylepgpv taqvvlqaai pltscgsspv pgttdghrpt aeapnttagq
    241 vpttevvgtt pgqaptaeps gttsvqvptt evistapvqm ptaestgmtp ekvpvsevmg
    301 ttlaemstpe atgmtpaevs ivvlsgttaa qvtttewvet tarelpipep egpdassims
    361 tesitgslgp lldgtatlrl vkrqvpldcv lyrygsfsvt ldivqgiesa eilqavpsge
    421 gdafeltvsc qgglpkeacm eisspgcqpp aqrlcqpvlp spacqlvlhq ilkggsgtyc
    481 lnvsladtns lavvstqlim pgqeaglgqv plivgillvl mavvlasliy rrrlmkqdfs
    541 vpqlphsssh wlrlprifcs cpigenspll sgqqv
    Premelanosome protein, isoform 3 preprotein NP_008859.1 (SEQ ID NO: 264)
    1 mdlvlkrcll hlavigalla vgatkvprnq dwlgvsrqlr tkawnrqlyp ewteaqrldc
    61 wrggqvslkv sndgptliga nasfsialnf pgsqkvlpdg qviwvnntii ngsqvwggqp
    121 vypqetddac ifpdggpcps gswsqkrsfv yvwktwgqyw qvlggpvsgl sigtgramlg
    181 thtmevtvyh rrgsrsyvpl ahsssaftit dqvpfsvsvs qlraldggnk hflrnqpltf
    241 alqlhdpsgy laeadlsytw dfgdssgtli sralvvthty lepgpvtaqv vlqaaiplts
    301 cgsspvpgtt dghrptaeap nttagqvptt evvgttpgqa ptaepsgtts vqvpttevis
    361 tapvqmptae stgmtpekvp vsevmgttla emstpeatgm tpaevsivvl sgttaaqvtt
    421 tewvettare lpipepegpd assimstesi tgslgplldg tatlrlvkrq vpldcvlyry
    481 gsfsvtldiv qgiesaeilq avpsgegdaf eltvscqggl pkeacmeiss pgcqppaqrl
    541 cqpvlpspac qlvlhqilkg gsgtyclnvs ladtnslavv stqlimpgqe aglgqvpliv
    601 gillvlmavv lasliyrrrl mkqdfsvpql phssshwlrl prifcscpig enspllsgqq
    661 v
    Premelanosome protein, isoform 4 preprotein NP_001307050.1 (SEQ ID NO: 265)
    1 mdlvlkrcll hlavigalla vgatkvprnq dwlgvsrqlr tkawnrqlyp ewteaqrldc
    61 wrggqvslkv sndgptliga nasfsialnf pgsqkvlpdg qviwvnntii ngsqvwggqp
    121 vypqetddac ifpdggpcps gswsqkrsfv yvwktwgqyw qvlggpvsgl sigtgramlg
    181 thtmevtvyh rrgsrsyvpl ahsssaftit dqvpfsvsvs qlraldggnk hflrnqpltf
    241 alqlhdpsgy laeadlsytw dfgdssgtli sralvvthty lepgpvtaqv vlqaaiplts
    301 cgsspvpgtt dghrptaeap nttagqvptt evvgttpgqa ptaepsgtts vqvpttevis
    361 tapvqmptae staaqvttte wvettarelp ipepegpdas simstesitg slgplldgta
    421 tlrlvkrqvp ldcvlyrygs fsvtldivqg iesaeilqav psgegdafel tvscqgglpk
    481 eacmeisspg cqppaqrlcq pvlpspacql vlhqilkggs gtyclnvsla dtnslavvst
    541 qlimpvpgil ltgqeaglgq vplivgillv lmavvlasli yrrrlmkqdf svpqlphsss
    601 hwlrlprifc scpigenspl lsgqqv
    Premelanosome protein, isoform 5 preprotein NP_001307051.1 (SEQ ID NO: 266)
    1 mdlvlkrcll hlavigalla vgatkvprnq dwlgvsrqlr tkawnrqlyp ewteaqrldc
    61 wrggqvslkv sndgptliga nasfsialnf pgsqkvlpdg qviwvnntii ngsqvwggqp
    121 vypqetddac ifpdggpcps gswsqkrsfv yvwktwgqyw qvlggpvsgl sigtgramlg
    181 thtmevtvyh rrgsrsyvpl ahsssaftit dqvpfsvsvs qlraldggnk hflrnqpltf
    241 alqlhdpsgy laeadlsytw dfgdssgtli sralvvthty lepgpvtaqv vlqaaiplts
    301 cgsspvpgtt dghrptaeap nttagqvptt evvgttpgqa ptaepsgtts vqvpttevis
    361 tapvqmptae staaqvttte wvettarelp ipepegpdas simstesitg slgplldgta
    421 tlrlvkrqvp ldcvlyrygs fsvtldivqg iesaeilqav psgegdafel tvscqgglpk
    481 eacmeisspg cqppaqrlcq pvlpspacql vlhqilkggs gtyclnvsla dtnslavvst
    541 qlimpgqeag lgqvplivgi llvlmavvla sliyrrrlmk qdfsvpqlph ssshwlrlpr
    601 ifcscpigen spllsgqqv
    Glutamate receptor ionotropic,NMDA 2A, isoform 1 precursor NP_000824.1,
    NP_001127879.1 (SEQ ID NO: 267)
    1 mgrvgywtll vlpallvwrg papsaaaekg ppalniavml ghshdvtere lrtlwgpeqa
    61 aglpldvnvv allmnrtdpk slithvcdlm sgarihglvf gddtdqeava qmldfissht
    121 fvpilgihgg asmimadkdp tstffqfgas iqqqatvmlk imqdydwhvf slvttifpgy
    181 refisfvktt vdnsfvgwdm qnvitldtsf edaktqvglk kihssvilly cskdeavlil
    241 searslgltg ydffwivpsl vsgntelipk efpsglisvs yddwdyslea rvrdgigilt
    301 taassmlekf syipeakasc ygqmerpevp mhtlhpfmvn vtwdgkdlsf teegyqvhpr
    361 lvvivinkdr ewekvgkwen htlslrhavw pryksfsdce pddnhlsivt leeapfvive
    421 didpltetcv rntvpcrkfv kinnstnegm nvkkcckgfc idilkklsrt vkftydlylv
    481 tngkhgkkvn nvwngmigev vyqravmavg sltineerse vvdfsvpfve tgisvmvsrs
    541 ngtvspsafl epfsasvwvm mfvmllivsa iavfvfeyfs pvgynrnlak gkaphgpsft
    601 igkaiwllwg lvfnnsvpvq npkgttskim vsvwaffavi flasytanla afmiqeefvd
    661 qvtglsdkkf qrphdysppf rfgtvpngst ernirnnypy mhqymtkfnq kgvedalvsl
    721 ktgkldafiy daavinykag rdegcklvti gsgyifattg ygialqkgsp wkrqidlall
    781 qfvgdgemee letlwltgic hneknevmss qldidnmagv fymlaaamal slitfiwehl
    841 fywklrfcft gvcsdrpgll fsisrgiysc ihgvhieekk kspdfnitgs qsnmlkllrs
    901 aknissmsnm nssrmdspkr aadfiqrgsl imdmvsdkgn lmysdnrsfq gkesifgdnm
    961 nelqtfvanr qkdnlnnyvf qgqhpltlne snpntvevav steskansrp rqlwkksvds
    1021 irqdslsqnp vsqrdeatae nrthslkspr ylpeemahsd isetsnratc hrepdnsknh
    1081 ktkdnfkrsv askypkdcse vertylktks ssprdkiyti dgekepgfhl dppqfvenvt
    1141 lpenvdfpdp yqdpsenfrk gdstlpmnrn plhneeglsn ndqyklyskh ftlkdkgsph
    1201 setseryrqn sthcrsclsn mptysghftm rspfkcdacl rmgnlydide dqmlqetgnp
    1261 atgeqvyqqd waqnnalqlq knklrisrqh sydnivdkpr eldlsrpsrs islkdrerll
    1321 egnfygslfs vpssklsgkk sslfpqgled skrsksllpd htsdnpflhs hrddqrlvig
    1381 rcpsdpykhs lpsqavndsy lrsslrstas ycsrdsrghn dvyisehvmp yaanknnmys
    1441 tprvlnscsn rrvykkmpsi esdv
    Glutamate receptor ionotropic,NMDA 2A, isoform 2 precursor NP_001127880.1
    (SEQ ID NO: 268)
    1 mgrvgywtll vlpallvwrg papsaaaekg ppalniavml ghshdvtere lrtlwgpeqa
    61 aglpldvnvv allmnrtdpk slithvcdlm sgarihglvf gddtdqeava qmldfissht
    121 fvpilgihgg asmimadkdp tstffqfgas iqqqatvmlk imqdydwhvf slvttifpgy
    181 refisfvktt vdnsfvgwdm qnvitldtsf edaktqvqlk kihssvilly cskdeavlil
    241 searslgltg ydffwivpsl vsgntelipk efpsglisvs yddwdyslea rvrdgigilt
    301 taassmlekf syipeakasc ygqmerpevp mhtlhpfmvn vtwdgkdlsf teegyqvhpr
    361 lvvivlnkdr ewekvgkwen htlslrhavw pryksfsdce pddnhlsivt leeapfvive
    421 didpltetcv rntvpcrkfv kinnstnegm nvkkcckgfc idilkklsrt vkftydlylv
    481 tngkhgkkvn nvwngmigev vyqravmavg sltineerse vvdfsvpfve tgisvmvsrs
    541 ngtvspsafl epfsasvwvm mfvmllivsa iavfvfeyfs pvgynrnlak gkaphgpsft
    601 igkaiwllwg lvfnnsvpvg npkgttskim vsvwaffavi flasytanla afmiqeefvd
    661 qvtglsdkkf qrphdysppf rfgtvpngst ernirnnypy mhqymtkfnq kgvedalvsl
    721 ktgkldafiy daavlnykag rdegcklvti gsgyifattg ygialqkgsp wkrqidlall
    781 qfvgdgemee letlwltgic hneknevmss qldidnmagv fymlaaamal slitfiwehl
    841 fywklrfcft gvcsdrpgll fsisrgiysc ihgvhieekk kspdfnltgs qsnmlkllrs
    901 aknissmsnm nssrmdspkr aadfiqrgsl imdmvsdkgn lmysdnrsfq gkesifgdnm
    961 nelqtfvanr qkdnlnnyvf qgqhpltlne snpntvevav steskansrp rqlwkksvds
    1021 irqdslsqnp vsqrdeatae nrthslkspr ylpeemahsd isetsnratc hrepdnsknh
    1081 ktkdnfkrsv askypkdcse vertylktks ssprdkiyti dgekepgfhl dppqfvenvt
    1141 lpenvdfpdp yqdpsenfrk gdstlpmnrn plhneeglsn ndqyklyskh ftlkdkgsph
    1201 setseryrqn sthcrsclsn mptysghftm rspfkcdacl rmgnlydide dgmlqetgmt
    1261 nawllgdapr tltntrchpr r
    Metabotropic glutamate receptor 3 precursor NP_000831.2 (SEQ ID NO: 269)
    1 mkmltrlqvl tlalfskgfl lslgdhnflr reikiegdlv lgglfpinek gtgteecgri
    61 nedrgiqrle amlfaidein kddyllpgvk lgvhildtcs rdtyaleqsl efvrasltkv
    121 deaeymcpdg syaiqenipl liagviggsy ssysiqvanl lrlfqipgis yastsaklsd
    181 ksrydyfart vppdfyqaka maeilrffnw tyvstvaseg dygetgieaf eqearlrnic
    241 iataekvgrs nirksydsvi rellqkpnar vvvlfmrsdd sreliaaasr anasftwvas
    301 dgwgaqesii kgsehvayga itlelasqpv rqfdryfqsl npynnhrnpw frdfweqkfq
    361 cslqnkrnhr rvcdkhlaid ssnyeqeski mfvvnavyam ahalhkmqrt lcpnttklcd
    421 amkildgkkl ykdyllkinf tapfnpnkda dsivkfdtfg dgmgrynvfn fqnvggkysy
    481 lkvghwaetl sldvnsihws rnsvptsqcs dpcapnemkn mqpgdvccwi cipcepyeyl
    541 adeftcmdcg sgqwptadlt gcydlpedyi rwedawaigp vtiaclgfmc tcmvvtvfik
    601 hnntplvkas grelcyillf gvglsycmtf ffiakpspvi calrrlglgs sfaicysall
    661 tktnciarif dgvkngaqrp kfispssqvf iclglilvqi vmvsvwlile apgtrrytla
    721 ekretvilkc nvkdssmlis ltydvilvil ctvyafktrk cpenfneakf igftmyttci
    781 iwlaflpify vtssdyrvqt ttmcisysls gfvvlgclfa pkvhiilfqp qknvvthrlh
    841 lnrfsvsgtg ttysqssast yvptvcngre vldsttssl
    HPV E6 concoprotein, NP_041325.1 (SEQ ID NO: 270)
    1 mhqkrtamfq dpqerprklp qlctelqtti hdiilecvyc kqqllrrevy dfafrdlciv
    61 yrdgnpyavc dkclkfyski seyrhycysl ygttleqqyn kplcdllirc incqkplcpe
    121 ekqrhldkkq rfhnirgrwt grcmsccrss rtrretql
    HPV E7 Oncoprotein, NP_041326.1 (SEQ ID NO: 271)
    1 mhgdtptlhe ymldlqpett dlycyeqlnd sseeedeidg pagqaepdra hynivtfcck
    61 cdstlrlcvq sthvdirtle dllmgtlgiv cpicsqkp
    GTPase HRas, isoform 1 NP_001123914.1, NP_005334.1 (SEQ ID NO: 272)
    1 mteyklvvvg aggvgksalt iqliqnhfvd eydptiedsy rkqvvidget clldildtag
    61 qeeysamrdq ymrtgegflc vfainntksf edihqyreqi krvkdsddvp mvlvgnkcdl
    121 aartvesrqa qdlarsygip yietsaktrq gvedafytlv reirqhklrk lnppdesgpg
    181 cmsckcvls
    GTPase HRas, isoform 3 NP_001304983.1 (SEQ ID NO: 273)
    1 mtcpwcwwgt svtwlhalwn lgrlrtspea tasptsrprp rpgraaalal apapgpsgtp
    61 rdpcdpaapr agvedafytl vreirqhklr klnppdesgp gcmsckcvls
    GTPase HRas, isoform 2 NP_789765.1 (SEQ ID NO: 274)
    1 mteyklvvvg aggvgksalt iqliqnhfvd eydptiedsy rkqvvidget clldildtag
    61 qeeysamrdq ymrtgegflc vfainntksf edihqyreqi krvkdsddvp mvlvgnkcdl
    121 aartvesrqa qdlarsygip yietsaktrq gsrsgsssss gtlwdppgpm
    Vascular endothelial growth factor receptor 2 precursor NP_002244.1 (SEQ ID
    NO: 275)
    1 mqskvllava lwlcvetraa svglpsvsld lprlsiqkdi ltikanttlq itcrgqrdld
    61 wlwpnnqsgs eqrvevtecs dglfcktlti pkvigndtga ykcfyretdl asviyvyvqd
    121 yrspfiasvs dqhgvvyite nknktvvipc lgsisnlnvs lcarypekrf vpdgnriswd
    181 skkgftipsy misyagmvfc eakindesyq simyivvvvg yriydvvlsp shgielsvge
    241 klvinctart elnvgidfnw eypsskhqhk klvnrdlktq sgsemkkfls tltidgvtrs
    301 dqglytcaas sglmtkknst fvrvhekpfv afgsgmeslv eatvgervri pakylgyppp
    361 eikwykngip lesnhtikag hvltimevse rdtgnytvil tnpiskekqs hvvslvvyvp
    421 pqigekslis pvdsyqygtt qtltctvyai ppphhihwyw qleeecanep sqavsvtnpy
    481 pceewrsved fqggnkievn knqfaliegk nktvstiviq aanvsalykc eavnkvgrge
    541 rvisfhvtrg peitlqpdmq pteqesvslw ctadrstfen ltwyklgpqp lpihvgelpt
    601 pvcknldtlw klnatmfsns tndilimelk naslqdqgdy vclaqdrktk krhcvvrqlt
    661 vlervaptit gnlenqttsi gesievscta sgnpppqimw fkdnetlved sgivlkdgnr
    721 nitirrvrke deglytcqac svlgcakvea ffiiegaqek tnleiiilvg taviamffwl
    781 llviilrtvk ranggelktg ylsivmdpde lpldehcerl pydaskwefp rdrlklgkpl
    841 grgafgqvie adafgidkta tcrtvavkml kegathsehr almselkili highhlnvvn
    901 llgactkpgg plmvivefck fgnlstylrs krnefvpykt kgarfrqgkd yvgaipvdlk
    961 rrldsitssq ssassgfvee kslsdveeee apedlykdfl tlehlicysf qvakgmefla
    1021 srkcihrdla arnillsekn vvkicdfgla rdiykdpdyv rkgdarlplk wmapetifdr
    1081 vytiqsdvws fgvllweifs lgaspypgvk ideefcrrlk egtrmrapdy ttpemyqtml
    1141 dcwhgepsqr ptfselvehl gnllqanaqq dgkdyivlpi setlsmeeds glslptspvs
    1201 cmeeeevcdp kfhydntagi sqylqnskrk srpvsvktfe dipleepevk vipddnqtds
    1261 gmvlaseelk tledrtklsp sfggmvpsks resvasegsn qtsgyqsgyh sddtdttvys
    1321 seeaellkli eigvqtgsta qilqpdsgtt lssppv
    Mast/stem cell growth acor receptor KIT, isoform 1 precursor NP_000213.1 (SEQ
    ID NO: 276)
    1 mrgargawdf lcvlllllrv qtgssqpsys pgepsppsih pgksdlivrv gdeirllctd
    61 pgfvkwtfei ldetnenkqn ewitekaeat ntgkytctnk hglsnsiyvf vrdpaklflv
    121 drslygkedn dtlvrcpltd pevtnyslkg cqgkplpkdl rfipdpkagi miksvkrayh
    181 rlclhcsvdq egksvlsekf ilkvrpafka vpvvsyskas yllregeeft vtctikdvss
    241 svystwkren sqtklqekyn swhhgdfnye rqatltissa rvndsgvfmc yanntfgsan
    301 vtttlevvdk gfinifpmin ttvfvndgen vdliveyeaf pkpehqqwiy mnrtftdkwe
    361 dypksenesn iryvselhlt rlkgteggty tflvsnsdvn aaiafnvyvn tkpeiltydr
    421 lvngmlqcva agfpeptidw yfcpgteqrc sasvlpvdvq tlnssgppfg klvvqssids
    481 safkhngtve ckayndvgkt sayfnfafkg nnkeqihpht lftplligfv ivagmmciiv
    541 miltykylqk pmyevqwkvv eeingnnyvy idptqlpydh kwefprnrls fgktlgagaf
    601 gkvveatayg liksdaamtv avkmlkpsah lterealmse lkvlsylgnh mnivnllgac
    661 tiggptlvit eyccygdlln flrrkrdsfi cskqedhaea alyknllhsk esscsdstne
    721 ymdmkpgvsy vvptkadkrr svrigsyier dvtpaimedd elaldledll sfsyqvakgm
    781 aflaskncih rdlaarnill thgritkicd fglardiknd snyvvkgnar lpvkwmapes
    841 ifncvytfes dvwsygiflw elfslgsspy pgmpvdskfy kmikegfrml spehapaemy
    901 dimktcwdad plkrptfkqi vqliekqise stnhiysnla ncspnrqkpv vdhsvrinsv
    961 gstasssqpl lvhddv
    Mast/stem cell growth acor receptor KIT, isoform 2 precursor NP_001087241.1
    (SEQ ID NO: 277)
    1 mrgargawdf lcvlllllrv qtgssqpsys pgepsppsih pgksdlivrv gdeirllctd
    61 pgfvkwtfei ldetnenkqn ewitekaeat ntgkytctnk hglsnsiyvf vrdpaklflv
    121 drslygkedn dtlvrcpltd pevtnyslkg cqgkplpkdl rfipdpkagi miksvkrayh
    181 rlclhcsvdq egksvlsekf ilkvrpafka vpvvsvskas yllregeeft vtctikdvss
    241 svystwkren sqtklqekyn swhhgdfnye rqatltissa rvndsgvfmc yanntfgsan
    301 vtttlevvdk gfinifpmin ttvfvndgen vdliveyeaf pkpehqqwiy mnrtftdkwe
    361 dypksenesn iryvselhlt rlkgteggty tflvsnsdvn aaiafnvyvn tkpeiltydr
    421 lvngmlqcva agfpeptidw yfcpgteqrc sasvlpvdvq tlnssgppfg klvvqssids
    481 safkhngtve ckayndvgkt sayfnfafke qihphtlftp lligfvivag mmciivmilt
    541 ykylqkpmye vqwkvveein gnnyvyidpt qlpydhkwef prnrlsfgkt lgagafgkvv
    601 eataygliks daamtvavkm lkpsahlter ealmselkvl sylgnhmniv nllgactigg
    661 ptiviteycc ygdllnflrr krdsficskq edhaeaalyk nllhskessc sdstneymdm
    721 kpgvsyvvpt kadkrrsvri gsyierdvtp aimeddelal dledllsfsy qvakgmafla
    781 skncihrdla arnillthgr itkicdfgla rdikndsnyv vkgnarlpvk wmapesifnc
    841 vytfesdvws ygiflwelfs lgsspypgmp vdskfykmik egfrmlspeh apaemydimk
    901 tcwdadplkr ptfkqivqli ekqisestnh iysnlancsp nrqkpvvdhs vrinsvgsta
    961 sssqpllvhd dv
    Plasma kallikrein isoform 1 preprotein NP_001639.1 (SEQ ID NO: 278)
    1 mwvpvvfltl svtwigaapl ilsrivggwe cekhsqpwqv lvasrgravc ggvlvhpqwv
    61 ltaahcirnk svillgrhsl fhpedtgqvf qvshsfphpl ydmsllknrf lrpgddsshd
    121 lmllrlsepa eltdavkvmd lptqepalgt tcyasgwgsi epeefltpkk lqcvdlhvis
    181 ndvcaqvhpq kvtkfmlcag rwtggkstcs gdsggplvcn gvlqgitswg sepcalperp
    241 slytkvvhyr kwikdtivan p
    Plasma kallikrein isoform 3 preprotein NP_001025218.1 (SEQ ID NO: 279)
    1 mwvpvvfltl svtwigaapl ilsrivggwe cekhsqpwqv lvasrgravc ggvlvhpqwv
    61 ltaahcirnk svillgrhsl fhpedtgqvf qvshsfphpl ydmsllknrf lrpgddsshd
    121 lmllrlsepa eltdavkvmd lptqepalgt tcyasgwgsi epeefltpkk lqcvdlhvis
    181 ndvcaqvhpq kvtkfmlcag rwtggkstcs wviliteltm palpmvlhgs lvpwrggv
    Plasma kallikrein isoform 4 preprotein NP_001025219.1 (SEQ ID NO: 280)
    1 mwvpvvfltl svtwigaapl ilsrivggwe cekhsqpwqv lvasrgravc ggvlvhpqwv
    61 ltaahcirkp gddsshdlml lrlsepaelt davkvmdlpt qepalgttcy asgwgsiepe
    121 efltpkklqc vdlhvisndv caqvhpqkvt kfmlcagrwt ggkstcsgds ggplvcngvl
    181 qgitswgsep calperpsly tkvvhyrkwi kdtivanp
    Tyrosine-protein kinase LCK, isoform a NP_001036236.1, NP_005347.3 (SEQ ID
    NO: 281)
    1 mgcgcsshpe ddwmenidvc enchypivpl dgkgtllirn gsevrdplvt yegsnppasp
    61 lqdnlvialh syepshdgdl gfekgeqlri leqsgewwka qslttgqegf ipfnfvakan
    121 slepepwffk nlsrkdaerq llapgnthgs fliresesta gsfslsvrdf dqnqgevvkh
    181 ykirnldngg fyispritfp glhelvrhyt nasdglctrl srpcqtqkpq kpwwedewev
    241 pretlklver lgagqfgevw mgyynghtkv avkslkqgsm spdaflaean lmkqlqhqrl
    301 vrlyavvtqe piyiiteyme ngslvdflkt psgikltink lldmaaqiae gmafieerny
    361 ihrdlraani lvsdtlscki adfglarlie dneytarega kfpikwtape ainygtftik
    421 sdvwsfgill teivthgrip ypgmtnpevi qnlergyrmv rpdncpeely qlmrlcwker
    481 pedrptfdyl rsvledffta tegqyqpqp
    Tyrosine-protein kinase LCK, isoform b NP_001317397.1 (SEQ ID NO: 282)
    1 mgcgcsshpe ddwmenidvc enchypivpl dgkgtllirn gsevrdplvt yegsnppasp
    61 lqdnlvialh syepshdgdl gfekgeqlri leqsgewwka qslttgqegf ipfnfvakan
    121 slepepwffk nlsrkdaerq llapgnthgs fliresesta gsfslsvrdf dqnqgevvkh
    181 ykirnldngg fyispritfp glhelvrhyt ryynghtkva vkslkqgsms pdaflaeanl
    241 mkqlqhqrlv rlyavvtqep iyiiteymen gslvdflktp sgikltinkl ldmaaqiaeg
    301 mafieernyi hrdlraanil vsdtlsckia dfglarlied neytaregak fpikwtapea
    361 inygtftiks dvwsfgillt eivthgripy pgmtnpeviq nlergyrmvr pdncpeelyq
    421 lmrlcwkerp edrptfdylr svledfftat egqyqpqp
    Legumain preprotein NP_001008530.1, NP_005597.3 (SEQ ID NO: 283)
    1 mvwkvavfls valgigavpi ddpedggkhw vvivagsngw ynyrhqadac hayqiihrng
    61 ipdeqivvmm yddiaysedn ptpgivinrp ngtdvyqgvp kdytgedvtp qnflavlrgd
    121 aeavkgigsg kvlksgpqdh vfiyftdhgs tgilvfpned lhvkdlneti hymykhkmyr
    181 kmvfyieace sgsmmnhlpd ninvyattaa npressyacy ydekrstylg dwysvnwmed
    241 sdvedltket lhkqyhlvks htntshvmqy gnktistmkv mqfqgmkrka sspvplppvt
    301 hldltpspdv pltimkrklm ntndleesrq lteeiqrhld arhlieksvr kivsllaase
    361 aeveqllser apltghscyp eallhfrthc fnwhsptyey alrhlyvlvn lcekpyplhr
    421 iklsmdhvol ghy
    Macrophage migration inhibitory factor NP_002406.1 (SEQ ID NO: 284)
    1 mpmfivntnv prasvpdgfl seltqqlaqa tgkppqyiav hvvpdqlmaf ggssepcalc
    61 slhsigkigg aqnrsyskll cgllaerlri spdrvyinyy dmnaanvgwn nstfa
    MAGE family member A1 NP_004979.3 (SEQ ID NO: 285)
    1 msleqrslhc kpeealeaqq ealglvcvqa atssssplvl gtleevptag stdppqspqg
    61 asafpttinf trqrqpsegs ssreeegpst scileslfra vitkkvadlv gflllkyrar
    121 epvtkaemle sviknykhcf peifgkases lqlvfgidvk eadptghsyv lvtclglsyd
    181 gllgdnqimp ktgfliivlv miamegghap eeeiweelsv mevydgrehs aygeprkllt
    241 qdlvqekyle yrqvpdsdpa ryeflwgpra laetsyvkvl eyvikvsarv rfffpslrea
    301 alreeeegv
    Melanoma-associated antigen 10 NP_001011543.2, NP_001238757.1, NP_066386.2
    (SEQ ID NO: 286)
    1 mprapkrqrc mpeedlqsqs etqglegaqa plaveedass ststsssfps sfpsssssss
    61 sscyplipst peevsaddet pnppqsaqia csspsvvasl pldqsdegss sqkeespstl
    121 qvlpdseslp rseidekvtd lvqfllfkyq mkepitkaei lesvirnyed hfpllfseas
    181 ecmllvfgid vkevdptghs fvlvtslglt ydgmlsdvqs mpktgilili lsiifiegyc
    241 tpeeviweal nmmglydgme hliygeprkl ltqdwvqeny leyrqvpgsd paryeflwgp
    301 rahaeirkms llkflakvng sdprsfplwy eealkdeeer aqdriattdd ttamasasss
    361 atgsfsype
    Melanoma-associated antigen 12 NP_001159858.1, NP_001159859.1, NP_005358.2
    (SEQ ID NO: 287)
    1 mpleqrsqhc kpeegleaqg ealglvgaqa pateeqetas ssstivevtl revpaaesps
    61 pphspqgast lpttinytlw sqsdegssne eqegpstfpd letsfqvals rkmaelvhfl
    121 llkyrarepf tkaemlgsvi rnfqdffpvi fskaseylql vfgievvevv righlyilvt
    181 clglsydgll gdnqivpktg lliivlaiia kegdcapeek iweelsvlea sdgredsvfa
    241 hprklltqdl vqenyleyrq vpgsdpacye flwgpralve tsyvkvlhhl lkisggphis
    301 ypplhewafr egee
    Melanoma-associated antigen 2 NP_001269430.1, NP_001269431.1, NP_001269433.1,
    NP_001269434.1, NP_005352.1, NP_786884.1, NP_786885.1 (SEQ ID NO: 288)
    1 mpleqrsqhc kpeeglearg ealglvgaqa pateeqqtas ssstlvevtl gevpaadsps
    61 pphspqgass fsttinytlw rqsdegssnq eeegprmfpd lesefqaais rkmvelvhfl
    121 llkyrarepv tkaemlesvl rncqdffpvi fskaseylql vfgievvevv pishlyilvt
    181 clglsydgll gdnqvmpktg lliivlaiia iegdcapeek iweelsmlev fegredsvfa
    241 hprkllmqdl vqenyleyrq vpgsdpacye flwgpralie tsyvkvlhht lkiggephis
    301 ypplheralr egee
    MAGE family member A3 NP_005353.1 (SEQ ID NO: 289)
    1 mpleqrsqhc kpeeglearg ealglvgaqa pateeqeaas ssstlvevtl gevpaaespd
    61 ppqspqgass lpttmnyplw sqsyedssnq eeegpstfpd lesefqaals rkvaelvhfl
    121 llkyrarepv tkaemlgsvv gnwqyffpvi fskassslql vfgielmevd pighlyifat
    181 clglsydgll gdnqimpkag lliivlaiia regdcapeek iweelsvlev fegredsilg
    241 dpkklltqhf vqenyleyrq vpgsdpacye flwgpralve tsyvkvlhhm vkisggphis
    301 ypplhewvlr egee
    Melanoma-associated antigen 4 NP_001011548.1, NP_001011549.1, NP_001011550.1,
    NP_002353.3 (SEQ ID NO: 290)
    1 msseqksqhc kpeegveaqe ealglvgaqa ptteeqeaav ssssplvpgt leevpaaesa
    61 gppqspqgas alpttisftc wrqpnegsss qeeegpstsp daeslfreal snkvdelahf
    121 llrkyrakel vtkaemlerv iknykrcfpv ifgkaseslk mifgidvkev dpasntytlv
    181 tclglsydgl lgnnqifpkt glliivlgti amegdsasee eiweelgvmg vydgrehtvy
    241 geprklltqd wvqenyleyr qvpgsnpary eflwgprala etsyvkvleh vvrvnarvri
    301 aypslreaal leeeegv
    Melanoma-associated antigen 6 NP_005354.1, NP_787064.1 (SEQ ID NO: 291)
    1 mpleqrsqhc kpeeglearg ealglvgaqa pateeqeaas ssstivevtl gevpaaespd
    61 ppgspqgass lpttmnyplw sqsyedssnq eeegpstfpd lesefqaals rkvaklvhfl
    121 llkyrarepv tkaemlgsvv gnwqyffpvi fskasdslql vfgielmevd pighvyifat
    181 clglsydgll gdnqimpktg fliiilaiia kegdcapeek iweelsvlev fegredsifg
    241 dpkklltqyf vqenyleyrq vpgsdpacye flwgpralie tsyvkvlhhm vkisggpris
    301 ypllhewalr egee
    Melanoma-associated antigen 9 NP_005356.1 (SEQ ID NO: 292)
    1 msleqrsphc kpdedleaqg edlglmgaqe ptgeeeetts ssdskeeevs aagsssppqs
    61 pqggasssis vyytlwsqfd egsssqeeee psssvdpaql efmfqealkl kvaelvhfll
    121 hkyrvkepvt kaemlesvik nykryfpvif gkasefmqvi fgtdvkevdp aghsyilvta
    181 lglscdsmlg dghsmpkaal liivlgvilt kdncapeevi wealsvmgvy vgkehmfyge
    241 prklltqdwv qenyleyrqv pgsdpahyef lwgskahaet syekvinylv mlnarepicy
    301 pslyeevlge eqegv
    Melanoma-associated antigen C2 NP_057333.1 (SEQ ID NO: 293)
    1 mppvpgvpfr nvdndsptsv eledwvdaqh ptdeeeeeas sasstlylvf spssfstsss
    61 lilggpeeee vpsgvipnit esipssppqg ppqgpsqspl ssccssfsws sfseesssqk
    121 gedtgtcqgl pdsessftyt ldekvaelve flllkyeaee pvteaemlmi vikykdyfpv
    181 ilkrarefme llfglaliev gpdhfcvfan tvgltdegsd degmpensll iiilsvifik
    241 gncaseeviw evlnavgvya grehfvygep relltkvwvq ghyleyrevp hssppyyefl
    301 wgprahsesi kkkvleflak lnntvpssfp swykdalkdv eervqatidt addatvmase
    361 slsvmssnvs fse
    Melanoma-associated antigen D1, isoform a NP_001005333.1 (SEQ ID NO: 294)
    1 maqkmdcgag llgfqnpdac ravchplpqp pastlplsaf ptlcdppysq lrdppavlsc
    61 yctplgaspa paeasvedsa llmqtlmeai qiseapptnq ataaaspqss qpptanemad
    121 iqvsaaaarp ksafkvqnat tkgpngvydf sqahnakdvp ntqpkaafks qnatpkgpna
    181 aydfsqaatt gelaanksem afkaqnattk vgpnatynfs qslnandlan srpktpfkaw
    241 ndttkaptad tqtqnvnqak matsqadiet dpgisepdga taqtsadgsq aqnlesrtii
    301 rgkrtrkinn lnveenssgd qrraplaagt wrsapvpvtt qnppgappnv lwqtplawqn
    361 psgwqnqtar qtpparqspp arqtppawqn pvawqnpviw pnpviwqnpv iwpnpivwpg
    421 pvvwpnplaw qnppgwqtpp gwqtppgwqg ppdwqgppdw plppdwplpp dwplptdwpl
    481 ppdwipadwp ippdwqnlrp spnlrpspns rasqnpgaaq prdvallqer anklvkylml
    541 kdytkvpikr semlrdiire ytdvypeiie racfvlekkf giqlkeidke ehlyilistp
    601 eslagilgtt kdtpklglll vilgvifmng nraseavlwe alrkmglrpg vrhpllgdlr
    661 klltyefvkq kyldyrrvpn snppeyeflw glrsyhetsk mkvlrfiaev qkrdprdwta
    721 qfmeaadeal daldaaaaea earaeartrm gigdeaysgp wswddiefel ltwdeegdfg
    781 dpwsripftf waryhqnars rfpqtfagpi igpggtasan faanfgaigf fwve
    Melanoma-associated antigen D1, isoform b NP_001005332.1, NP_008917.3 (SEQ ID
    NO: 295)
    1 maqkmdcgag llgfqaeasv edsallmqtl meaiqiseap ptnqataaas pqssqpptan
    61 emadiqvsaa aarpksafkv qnattkgpng vydfsqahna kdvpntqpka afksqnatpk
    121 gpnaaydfsq aattgelaan ksemafkaqn attkvgpnat ynfsqslnan dlansrpktp
    181 fkawndttka ptadtqtqnv nqakmatsqa dietdpgise pdgataqtsa dgsqaqnles
    241 rtiirgkrtr kinnlnveen ssgdqrrapl aagtwrsapv pvttqnppga ppnvlwqtpl
    301 awqnpsgwqn qtarqtppar qspparqtpp awqnpvawqn pviwpnpviw qnpviwpnpi
    361 vwpgpvvwpn plawqnppgw qtppgwqtpp gwqgppdwqg ppdwplppdw plppdwplpt
    421 dwplppdwip adwpippdwq nlrpspnlrp spnsrasqnp gaaqprdval lqeranklvk
    481 ylmlkdytkv pikrsemlrd iireytdvyp eiieracfvl ekkfgiqlke idkeehlyil
    541 istpeslagi lgttkdtpkl glllvilgvi fmngnrasea vlwealrkmg lrpgvrhpll
    601 gdlrklltye fvkqkyldyr rvpnsnppey eflwglrsyh etskmkvlrf iaevqkrdpr
    661 dwtaqfmeaa dealdaldaa aaeaearaea rtrmgigdea vsgpwswddi efelltwdee
    721 gdfgdpwsri pftfwaryhq narsrfpqtf agpiigpggt asanfaanfg aigffwve
    Mitogen-activated protein kinase kinase kinase 5 NP_005914.1 (SEQ ID NO: 296)
    1 msteadegit fsvppfapsg fctipeggic rrggaaavge geehqlpppp pgsfwnvesa
    61 aapgigcpaa tssssatrgr gssvgggsrr ttvayvinea sqgqlvvaes ealqslreac
    121 etvgatletl hfgkldfget tvldrfynad iavvemsdaf rqpslfyhlg vresfsmann
    181 iilycdtnsd slqslkeiic qkntmctgny tfvpymitph nkvyccdssf mkgltelmqp
    241 nfelllgpic lplvdrfiql lkvaqasssq yfresilndi rkarnlytgk elaaelarir
    301 qrvdnievlt adivinllls yrdiqdydsi vklvetlekl ptfdlashhh vkfhyafaln
    361 rrnlpgdrak aldimipmvq segqvasdmy clvgriykdm fldsnftdte srdhgaswfk
    421 kafeseptlq sginyavlll aaghqfessf elrkvgvkls sllgkkgnle klqsywevgf
    481 flgasvland hmrviqasek lfklktpawy lksivetili ykhfvkltte qpvakqelvd
    541 fwmdflveat ktdvtvvrfp vlileptkiy gpsylsinne veektisiwh vlpddkkgih
    601 ewnfsassvr gvsiskfeer ccflyvlhns ddfqiyfcte lhckkffemv ntiteekgrs
    661 teegdcesdl leydyeyden gdrvvlgkgt ygivyagrdl snqvriaike iperdsrysq
    721 plheeialhk hlkhknivqy lgsfsengfi kifmeqvpgg slsallrskw gplkdneqti
    781 gfytkqileg lkylhdnqiv hrdikgdnvl intysgvlki sdfgtskrla ginpctetft
    841 gtlqymapei idkgprgygk aadiwslgct iiematgkpp fyelgepqaa mfkvgmfkvh
    901 peipesmsae akafilkcfe pdpdkracan dllvdeflkv sskkkktqpk lsalsagsne
    961 ylrsislpvp vlvedtssss eygsyspdte lkvdpfsfkt rakscgerdv kgirtlflgi
    1021 pdenfedhsa ppspeekdsg ffmlrkdser ratlhrilte dqdkivrnlm eslaqgaeep
    1081 klkwehittl iaslrefvrs tdrkiiattl sklkleldfd shgisqvqvv lfgfqdavnk
    1141 vlrnhnikph wmfaldsiir kavqtaitil vpelrphfsl asesdtadqe dldveddhee
    1201 qpsnqtvrrp qaviedavat sgvstlsstv shdsqsahrs lnvqlgrmki etnrlleelv
    1261 rkekelqall hraieekdqe ikhlklksqp ieipelpvfh lnssgtnted seltdwlrvn
    1321 gadedtisrf laedytlldv lyyvtrddlk clrlrggmlc tlwkaiidfr nkqt
    Mitogen-activated protein kinase kinase kinase 9, isoform 1 NP_149132.2 (SEQ
    ID NO: 297)
    1 mepsrallgc lasaaaaapp gedgagagae eeeeeeeeaa aavgpgelgc daplpywtav
    61 feyeaagede ltlrlgdvve vlskdsqvsg degwwtgqln qrvgifpsny vtprsafssr
    121 cqpggedpsc yppiqlleid faeltleeii giggfgkvyr afwigdevav kaarhdpded
    181 isqtienvrq eaklfamlkh pniialrgvc lkepnlclvm efarggpinr vlsgkrippd
    241 ilvnwavqia rgmnylhdea ivpiihrdlk ssnililqkv engdlsnkil kitdfglare
    301 whrttkmsaa gtyawmapev irasmfskgs dvwsygvllw elltgevpfr gidglavayg
    361 vamnklalpi pstcpepfak lmedcwnpdp hsrpsftnil dqlttieesg ffempkdsfh
    421 clqdnwkhei qemfdqlrak ekelrtweee ltraalqqkn qeellrrreq elaereidil
    481 erelniiihq lcqekprvkk rkgkfrksrl klkdgnrisl psdfqhkftv qasptmdkrk
    541 slinsrsspp asptiiprlr aiqltpgess ktwgrssvvp keegeeeekr apkkkgrtwg
    601 pgtlgqkela sgdegspqrr ekanglstps esphfhlglk slvdgykqws ssapnlvkgp
    661 rsspalpgft slmemallaa swvvpidiee dedsegpgsg esrlqhspsq sylcipfprg
    721 edgdgpssdg iheeptpvns atstpqltpt nslkrggahh rrcevallgc gavlaatglg
    781 fdlleagkcq llpleepepp areekkrreg lfqrssrprr stsppsrklf kkeepmlllg
    841 dpsasltlls lssisecnst rsllrsdsde ivvyempvsp veapplspct hnplvnvrve
    901 rfkrdpnqsl tpthvtlttp sqpsshrrtp sdgalkpetl lasrspssng lspspgagml
    961 ktpspsrdpg efprlpdpnv vfpptprrwn tqqdstlerp ktleflprpr psanrqrldp
    1021 wwfvspshar stspanssst etpsnldscf asssstveer pglpallpfq agplpptert
    1081 lldldaegqs qdstvplcra elnthrpapy eiqqefws
    Mitogen-activated protein kinase kinase kinase 9, isoform 2 NP_001271159.1
    (SEQ ID NO: 298)
    1 mepsrallgc lasaaaaapp gedgagagae eeeeeeeeaa aavgpgelgc daplpywtav
    61 feyeaagede ltlrlgdvve vlskdsqvsg degwwtgqln qrvgifpsny vtprsafssr
    121 cqpggedpsc yppiqlleid faeltleeii giggfgkvyr afwigdevav kaarhdpded
    181 isqtienvrq eaklfamlkh pniialrgvc lkepnlclvm efarggplnr vlsgkrippd
    241 ilvnwavqia rgmnylhdea ivpiihrdlk ssnililqkv engdlsnkil kitdfglare
    301 whrttkmsaa gtyawmapev irasmfskgs dvwsygvllw elltgevpfr gidglavayg
    361 vamnklalpi pstcpepfak lmedcwnpdp hsrpsftnil dqlttieesg ffempkdsfh
    421 clqdnwkhei qemfdqlrak ekelrtweee ltraalqqkn geellrrreq elaereidil
    481 erelniiihq lcqekprvkk rkgkfrksrl klkdgnrisl psdfqhkftv qasptmdkrk
    541 slinsrsspp asptiiprlr aiqltpgess ktwgrssvvp keegeeeekr apkkkgrtwg
    601 pgtlgqkela sgdegspqrr ekanglstps esphfhlglk slvdgykqws ssapnlvkgp
    661 rsspalpgft slmemededs egpgsgesrl qhspsqsylc ipfprgedgd gpssdgihee
    721 ptpvnsatst pqltptnslk rggahhrrce vallgcgavl aatglgfdll eagkcqllpl
    781 eepepparee kkrreglfqr ssrprrstsp psrklfkkee pmlllgdpsa sltllslssi
    841 secnstrsll rsdsdeivvy empvspveap plspcthnpl vnvrverfkr dpnqsltpth
    901 vtlttpsqps shrrtpsdga lkpetllasr spssnglsps pgagmlktps psrdpgefpr
    961 lpdpnvvfpp tprrwntqqd stlerpktle flprprpsan rqrldpwwfv spsharstsp
    1021 anssstetps nldscfasss stveerpglp allpfqagpl pptertlldl daegqsqdst
    1081 vplcraelnt hrpapyeiqq efws
    Mitogen-activated protein kinase kinase kinase 9, isoform 3 NP_001271160.1
    (SEQ ID NO: 299)
    1 meltgleval vlilqkveng dlsnkilkit dfglarewhr ttkmsaagty awmapevira
    61 smfskgsdvw sygvllwell tgevpfrgid glavaygvam nklalpipst cpepfaklme
    121 dcwnpdphsr psftnildql ttieesgffe mpkdsfhclq dnwkheiqem fdqlrakeke
    181 lrtweeeltr aalqqknqee llrrreqela ereidilere lniiihqlcq ekprvkkrkg
    241 kfrksrlklk dgnrislpsd fqhkftvqas ptmdkrksli nsrssppasp tiiprlraiq
    301 cetvsqiswg qntqghlspa lsshrlvqac sihnfchlss tmciymhilt pgessktwgr
    361 ssvvpkeege eeekrapkkk grtwgpgtlg qkelasgdeg lkslvdgykq wsssapnlvk
    421 gprsspalpg ftslmemall aaswvvpidi eededsegpg sgesrlqhsp sqsylcipfp
    481 rgedgdgpss dgiheeptpv nsatstpqlt ptnslkrgga hhrrcevall gcgavlaatg
    541 lgfdlleagk cqllpleepe ppareekkrr eglfqrssrp rrstsppsrk lfkkeepmll
    601 lgdpsasltl lslssisecn strsllrsds deivvyempv spveapplsp cthnplvnvr
    661 verfkrdpnq sltpthvtlt tpsqpsshrr tpsdgalkpe tllasrspss nglspspgag
    721 mlktpspsrd pgefprlpdp nvvfpptprr wntqqdstle rpktleflpr prpsanrqrl
    781 dpwwfvspsh arstspanss stetpsnlds cfasssstve erpglpallp fqagplppte
    841 rtlldldaeg qsqdstvplc raelnthrpa pyeiqqefws
    Mitogen-activated protein kinase kinase kinase 9, isoform 4 NP_001271161.1
    (SEQ ID NO: 300)
    1 msaagtyawm apevirasmf skgsdvwsyg vllwelltge vpfrgidgla vaygvamnkl
    61 alpipstcpe pfaklmedcw npdphsrpsf tnildqltti eesgffempk dsfhclqdnw
    121 kheiqemfdq lrakekelrt weeeltraal qqknqeellr rreqelaere idilerelni
    181 iihqlcqekp rvkkrkgkfr ksrlklkdgn rislpsdfqh kftvqasptm dkrkslinsr
    241 ssppasptii prlraiqcet vsgiswgqnt qghlspalss hrlvqacsih nfchlsstmc
    301 iymhiltpge ssktwgrssv vpkeegeeee krapkkkgrt wgpgtlggke lasgdeglks
    361 lvdgykqwss sapnlvkgpr sspalpgfts lmemallaas wvvpidieed edsegpgsge
    421 srlqhspsqs ylcipfprge dgdgpssdgi heeptpvnsa tstpqltptn slkrggahhr
    481 rcevallgcg avlaatglgf dlleagkcql lpleepeppa reekkrregl fqrssrprrs
    541 tsppsrklfk keepmlllgd psasltllsl ssisecnstr sllrsdsdei vvyempvspv
    601 eapplspcth nplvnvrver fkrdpnqslt pthvtlttps qpsshrrtps dgalkpetll
    661 asrspssngl spspgagmlk tpspsrdpge fprlpdpnvv fpptprrwnt qqdstlerpk
    721 tleflprprp sanrqrldpw wfvspshars tspanssste tpsnldscfa sssstveerp
    781 glpallpfqa gplpptertl ldldaeggsq dstvplcrae lnthrpapye iqqefws
    Mitogen-activated protin kinase 1 NP_002736.3, NP_620407.1 (SEQ ID NO: 301)
    1 maaaaaagag pemvrgqvfd vgprytnlsy igegaygmvc saydnvnkvr vaikkispfe
    61 hqtycqrtlr eikillrfrh eniigindii raptieqmkd vyivqdlmet dlykllktqh
    121 lsndhicyfl yqilrglkyi hsanvlhrdl kpsnlllntt cdlkicdfgl arvadpdhdh
    181 tgflteyvat rwyrapeiml nskgytksid iwsvgcilae mlsnrpifpg khyldqlnhi
    241 lgilgspsqe dlnciinlka rnyllslphk nkvpwnrlfp nadskaldll dkmltfnphk
    301 rieveqalah pyleqyydps depiaeapfk fdmelddlpk eklkelifee tarfqpgyrs
    Melan-A NP_005502.1 (SEQ ID NO: 302)
    1 mpredahfiy gypkkghghs yttaeeaagi giltvilgvl lligcwycrr rngyralmdk
    61 slhvgtqcal trrcpqegfd hrdskvslqe kncepvvpna ppayeklsae qspppysp
    Melanotransferrin, isoform 1 preprotein NP_005920.2 (SEQ ID NO: 303)
    1 mrgpsgalwl llalrtvlgg mevrwcatsd peqhkcgnms eafreagiqp sllcvrgtsa
    61 dhcvqliaaq eadaitldgg aiyeagkehg lkpvvgevyd qevgtsyyav avvrrsshvt
    121 idtlkgvksc htginrtvgw nvpvgylves grlsvmgcdv lkaysdyfgg scvpgagets
    181 yseslcrlcr gdssgegvcd kspleryydy sgafrclaeg agdvafvkhs tvlentdgkt
    241 lpswgqalls qdfellcrdg sradvtewrq chlarvpaha vvvradtdgg lifrllnegq
    301 rlfshegssf qmfsseaygq kdllfkdsts elvpiatqty eawlgheylh amkgllcdpn
    361 rlppylrwcv lstpeiqkcg dmavafrrqr lkpeiqcvsa kspqhcmeri qaeqvdavtl
    421 sgediytagk tyglvpaage hyapedssns yyvvavvrrd sshaftldel rgkrschagf
    481 gspagwdvpv galiqrgfir pkdcdvltav seffnascvp vnnpknypss lcalcvgdeq
    541 grnkcvgnsq eryygyrgaf rclvenagdv afvrhttvfd ntnghnsepw aaelrsedye
    601 llcpngarae vsqfaacnla qipphavmvr pdtniftvyg lldkaqdlfg ddhnkngfkm
    661 fdssnyhgqd llfkdatvra vpvgekttyr gwlgldyvaa legmssqqcs gaaapapgap
    721 llplllpala arllppal
    Melanotransferrin, isoform 2 precursor NP_201573.1 (SEQ ID NO: 304)
    1 mrgpsgalwl llalrtvlgg mevrwcatsd peqhkcgnms eafreagiqp sllcvrgtsa
    61 dhcvqliaaq eadaitldgg aiyeagkehg lkpvvgevyd qevgtsyyav avvrrsshvt
    121 idtlkgvksc htginrtvgw nvpvgylves grlsvmgcdv lkavsdyfgg scvpgagets
    181 yseslcrlcr gdssgegvcd kspleryydy sgafrclaeg agdvafvkhs tvlentdesp
    241 srrqtwtrse eeegecpahe earrtmrssa gqawkwapvh rpqdesdkge fgkraksrdm
    301 lg
    Baculoviral IAP repeat containing 7, isoform alpha NP_647478.1 (SEQ ID NO:
    305)
    1 mgpkdsakcl hrgpqpshwa agdgptqerc gprslgspvl gldtcrawdh vdgqilgqlr
    61 plteeeeeeg agatlsrgpa fpgmgseelr lasfydwplt aevppellaa agffhtghqd
    121 kvrcffcygg lqswkrgddp wtehakwfps cqfllrskgr dfvhsvgeth sqllgswdpw
    181 eepedaapva psvpasgype lptprrevqs esaqepggvs paeaqrawwv leppgardve
    241 aqlrrlqeer tckvcldrav sivfvpcghl vcaecapglq lcpicrapvr srvrtfls
    Baculoviral IAP repeat containing 7, isoform beta NP_071444.1 (SEQ ID NO:
    306)
    1 mgpkdsakcl hrgpqpshwa agdgptqerc gprslgspvl gldtcrawdh vdgqilgqlr
    61 plteeeeeeg agatlsrgpa fpgmgseelr lasfydwplt aevppellaa agffhtghqd
    121 kvrcffcygg lqswkrgddp wtehakwfps cqfllrskgr dfvhsvqeth sqllgswdpw
    181 eepedaapva psvpasgype lptprrevqs esaqepgard veaqlrrlqe ertckvcldr
    241 avsivfvpcg hlvcaecapg lqlcpicrap vrsrvrtfls
    Neutrophil collagenase, isoform 1 preprotein NP_002415.1 (SEQ ID NO: 307)
    1 mfslktlpfl lllhvqiska fpvsskeknt ktvqdylekf yqlpsnqyqs trkngtnviv
    61 eklkemqrff glnvtgkpne etldmmkkpr cgvpdsggfm ltpgnpkwer tnityrirny
    121 tpqlseaeve raikdafelw svaspliftr isqgeadini afyqrdhgdn spfdgpngil
    181 ahafqpgqgi ggdahfdaee twtntsanyn lflvaahefg hslglahssd pgalmypnya
    241 fretsnyslp qddidgiqai yglssnpiqp tgpstpkpcd psltfdaitt lrgeilffkd
    301 ryfwrrhpql qrvemnfisl fwpslptgiq aayedfdrdl iflfkgnqyw alsgydilqg
    361 ypkdisnygf pssvqaidaa vfyrsktyff vndqfwrydn qrqfmepgyp ksisgafpgi
    421 eskvdavfqq ehffhvfsgp ryyafdliaq rvtrvargnk wlncryg
    Neutrophil collagenase, isoform 2 NP_001291370.1, NP_001291371.1 (SEQ ID NO:
    308)
    1 mqqipqeksi ndylekfyql psnqyqstrk ngtnvivekl kemqrffgln vtgkpneetl
    61 dmmkkprcgv pdsggfmltp gnpkwertnl tyrirnytpq lseaeverai kdafelwsva
    121 spliftrisq geadiniafy qrdhgdnspf dgpngilaha fqpgqgiggd ahfdaeetwt
    181 ntsanynlfl vaahefghsl glahssdpga lmypnyafre tsnyslpqdd idgiqaiygl
    241 ssnpiqptgp stpkpcdpsl tfdaittlrg eilffkdryf wrrhpqlqrv emnfislfwp
    301 slptgiqaay edfdrdlifl fkgnqywals gydilqgypk disnygfpss vqaidaavfy
    361 rsktyffvnd qfwrydnqrq fmepgypksi sgafpgiesk vdavfqqehf fhvfsgpryy
    421 afdliaqrvt rvargnkwln cryg
    Mesothelin, isoform 1 preprotein NP_001170826.1, NP_005814.2 (SEQ ID NO: 309)
    1 malptarpll gscgtpalgs llfllfslgw vqpsrtlage tgqeaapldg vlanppniss
    61 lsprqllgfp caevsglste rvrelavala qknvklsteq lrclahrlse ppedldalpl
    121 dlllflnpda fsgpqactrf fsritkanvd llprgaperq rllpaalacw gvrgsllsea
    181 dvralgglac dlpgrfvaes aevllprlvs cpgpldqdqq eaaraalqgg gppygppstw
    241 systmdalrg llpvlgqpii rsipqgivaa wrqrssrdps wrqpertilr prfrrevekt
    301 acpsgkkare ideslifykk weleacvdaa llatqmdrvn aipftyeqld vlkhkldely
    361 pqgypesviq hlgylflkms pedirkwnvt sletlkalle vnkghemspq vatlidrfvk
    421 grgqldkdtl dtltafypgy lcslspeels svppssiwav rpqdldtcdp rqldvlypka
    481 rlafqnmngs eyfvkiqsfl ggaptedlka lsqqnvsmdl atfmklrtda vlpltvaevq
    541 kllgphvegl kaeerhrpvr dwilrqrqdd ldtlglglqg gipngylvld lsmqealsgt
    601 pcllgpgpvl tvlalllast la
    Mesothelin, isoform 2 preprotein NP_037536.2 (SEQ ID NO: 310)
    1 malptarpll gscgtpalgs llfllfslgw vqpsrtlage tgqeaapldg vlanppniss
    61 lsprqllgfp caevsglste rvrelavala qknvklsteq lrclahrlse ppedldalpl
    121 dlllflnpda fsgpqactrf fsritkanvd llprgaperq rllpaalacw gvrgsllsea
    181 dvralgglac dlpgrfvaes aevllprlvs cpgpldqdqq eaaraalqgg gppygppstw
    241 systmdalrg llpvlgqpii rsipqgivaa wrqrssrdps wrqpertilr prfrrevekt
    301 acpsgkkare ideslifykk weleacvdaa llatqmdrvn aipftyeqld vlkhkldely
    361 pqgypesviq hlgylflkms pedirkwnvt sletlkalle vnkghemspq aprrplpqva
    421 tlidrfvkgr gqldkdtldt ltafypgylc slspeelssv ppssiwavrp qdldtcdprq
    481 ldvlypkarl afqnmngsey fvkiqsflgg aptedlkals qqnvsmdlat fmklrtdavl
    541 pltvaevqkl lgphveglka eerhrpvrdw ilrqrqddld tlglglqggi pngylvldls
    601 mqealsgtpc llgpgpvltv lalllastla
    Mucin-1, isoform 1 precursor NP_002447.4 (SEQ ID NO: 311)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knalstgvsf
    61 fflsfhisnl qfnssledps tdyyqelqrd isemflqiyk qggflglsni kfrpgsvvvq
    121 ltlafregti nvhdvetqfn qykteaasry nltisdvsvs dvpfpfsaqs gagvpgwgia
    181 llvlvcvlva laivyliala vcqcrrknyg qldifpardt yhpmseypty hthgryvpps
    241 stdrspyekv sagnggssls ytnpavaats anl
    Mucin-1, isoform 2 precursor NP_001018016.1 (SEQ ID NO: 312)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 nafnssledp stdyyqelqr disemflqiy kqggflglsn ikfrpgsvvv qltlafregt
    121 invhdvetqf nqykteaasr ynitisdvsv sdvpfpfsaq sgagvpgwgi allvlvcvlv
    181 alaivylial avcqcrrkny gqldifpard tyhpmseypt yhthgryvpp sstdrspyek
    241 vsagnggssl sytnpavaat sanl
    Mucin-1, isoform 3 precursor NP_001018017.1 (SEQ ID NO: 313)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knafnssled
    61 pstdyyqelq rdisemflqi ykqggflgls nikfrpgsvv vqltlafreg tinvhdvetq
    121 fnqykteaas rynltisdvs vsdvpfpfsa qsgagvpgwg iallvlvcvl valaivylia
    181 lavcqcrrkn ygqldifpar dtyhpmseyp tyhthgryvp psstdrspye kvsagnggss
    241 lsytnpavaa tsanl
    Mucin-1, isoform 5 precursor NP_001037855.1 (SEQ ID NO: 314)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knaipapttt
    61 kscretflkc fcrfinkgvf waspilssvs dvpfpfsaqs gagvpgwgia llvlvcvlva
    121 laivyliala vcgcrrknyg qldifpardt yhpmseypty hthgryvpps stdrspyekv
    181 sagnggssls ytnpavaats anl
    Mucin-1, isoform 6 precursor NP_001037856.1 (SEQ ID NO: 315)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knafnssled
    61 pstdyyqelq rdisemavcq crrknygqld ifpardtyhp mseyptyhth gryvppsstd
    121 rspyekvsag nggsslsytn pavaatsanl
    Mucin-1, isoform 7 precursor NP_001037857.1 (SEQ ID NO: 316)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 nafnssledp stdyyqelqr disemavcqc rrknygqldi fpardtyhpm seyptyhthg
    121 ryvppsstdr spyekvsagn ggsslsytnp avaatsanl
    Mucin-1, isoform 8 precursor NP_001037858.1 (SEQ ID NO: 317)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knaipapttt
    61 kscretflkc fcrfinkgvf waspilssvw gwgarlghra agaglcsgca ghclshclgc
    121 lsvppkelra aghlsspgyl psyervphlp hpwalcap
    Mucin-1, isoform 9 precursor NP_001191214.1 (SEQ ID NO: 318)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knaysmtssv
    61 lsshspgsgs sttqgqdvtl apatepasgs aatwgqdvts vpvtrpalgs ttppandvts
    121 apdnkpapgs tappahgvts apdtrpapgs tappahgvts apdnrpalgs tappvhnvts
    181 asgsasgsas tlvhngtsar atttpaskst pfsipshhsd tpttlashst ktdassthhs
    241 tvppltssnh stspqlstgv sffflsfhis nlqfnssled pstdyyqelq rdisemflqi
    301 ykqggflgls nikfrpgsvv vqltlafreg tinvhdvetq fnqykteaas rynitisdvs
    361 vsdvpfpfsa qsgagvpgwg iallvlvcvl valaivylia lavcqcrrkn ygqldifpar
    421 dtyhpmseyp tyhthgryvp psstdrspye kvsagnggss lsytnpavaa tsanl
    Mucin-1, isoform 10 precursor NP_001191215.1 (SEQ ID NO: 319)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 naysmtssvl sshspgsgss ttqgqdvtla patepasgsa atwgqdvtsv pvtrpalgst
    121 tppandvtsa pdnkpapgst appahgvtsa pdtrpapgst appahgvtsa pdnrpalgst
    181 appvhnvtsa sgsasgsast lvhngtsara tttpaskstp fsipshhsdt pttlashstk
    241 tdassthhst vppltssnhs tspqlstgvs ffflsfhisn lqfnssledp stdyyqelqr
    301 disemflqiy kqggflglsn ikfrpgsvvv qltlafregt invhdvetqf nqykteaasr
    361 ynltisdvsv sdvpfpfsaq sgagvpgwgi allvlvcvlv alaivylial avcqcrrkny
    421 gqldifpard tyhpmseypt yhthgryvpp sstdrspyek vsagnggssl sytnpavaat
    481 sanl
    Mucin-1, isoform 11 precursor NP_001191216.1 (SEQ ID NO: 320)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 nalstgvsff flsfhisnlq fnssledpst dyyqelqrdi semflqiykq ggflglsnik
    121 frpgsvvvql tlafregtin vhdvetqfnq ykteaasryn ltisdvsysd vpfpfsaqsg
    181 agvpgwgial lvlvcvlval aivylialav cqcrrknygq ldifpardty hpmseyptyh
    241 thgryvppss tdrspyekvs agnggsslsy tnpavaatsa nl
    Mucin-1, isoform 12 precursor NP_001191217.1 (SEQ ID NO: 321)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 nafnssledp stdyygelqr disemflqiy kqggflglsn ikfrpgsvvv qltlafregt
    121 invhdvetqf nqykteaasr ynitisdvsv wgwgarlghr aagaglcsgc aghclshclg
    181 clsvppkelr aaghlsspgy lpsyervphl phpwalcap
    Mucin-1, isoform 13 precursor NP_001191218.1 (SEQ ID NO: 322)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 naiykqggfl glsnikfrpg svvvqltlaf regtinvhdv etqfnqykte aasrynltis
    121 dvsysdvpfp fsaqsgagvp gwgiallvlv cvlvalaivy lialavcqcr rknygqldif
    181 pardtyhpms eyptyhthgr yvppsstdrs pyekvsagng gsslsytnpa vaatsanl
    Mucin-1, isoform 14 precursor NP_001191219.1 (SEQ ID NO: 323)
    1 mtpgtqspff llllltvltg geketsatqr ssvpsstekn aiykqggflg lsnikfrpgs
    61 vvvqltlafr egtinvhdve tqfnqyktea asrynltisd vsysdvpfpf saqsgagvpg
    121 wgiallvlvc vlvalaivyl ialavcqcrr knygqldifp ardtyhpmse yptyhthgry
    181 vppsstdrsp yekvsagngg sslsytnpav aatsanl
    Mucin-1, isoform 15 precursor NP_001191220.1 (SEQ ID NO: 324)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 naflqiykqg gflglsnikf rpgsvvvqlt lafregtinv hdvetqfnqy kteaasrynl
    121 tisdvsysdv pfpfsaqsga gvpgwgiall vlvcvlvala ivylialavc qcrrknygql
    181 difpardtyh pmseyptyht hgryvppsst drspyekvsa gnggsslsyt npavaatsan
    241 l
    Mucin-1, isoform 16 precursor NP_001191221.1 (SEQ ID NO: 325)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 naipaptttk scretflkwp gsvvvqltla fregtinvhd vetqfnqykt eaasryniti
    121 sdvsysdvpf pfsaqsgagv pgwgiallvl vcvlvalaiv ylialavcqc rrknygqldi
    181 fpardtyhpm seyptyhthg ryvppsstdr spyekvsagn ggsslsytnp avaatsanl
    Mucin-1, isoform 17 precursor NP_001191222.1 (SEQ ID NO: 326)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knalstgvsf
    61 fflsfhisnl qfnssledps tdyyqelqrd isemflqiyk qggflglsni kfrpgsvvvq
    121 ltlafregti nvhdvetqfn qykteaasry nitisdvsgc lsvppkelra aghlsspgyl
    181 psyervphlp hpwalcap
    Mucin-1, isoform 18 precursor NP_001191223.1 (SEQ ID NO: 327)
    1 mtpgtqspff llllltvltv vtgsghasst pggeketsat qrssvpsste knaipapttt
    61 kscretflkw pgsvvvqltl afregtinvh dvetqfnqyk teaasrynit isdvsysdvp
    121 fpfsaqsgag vpgwgiallv lvcvlvalai vylialavcq crrknygqld ifpardtyhp
    181 mseyptyhth gryvppsstd rspyekvsag nggsslsytn pavaatsanl
    Mucin-1, isoform 19 precursor NP_001191224.1 (SEQ ID NO: 328)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 nafnssledp stdyyqelqr disemsgagv pgwgiallvl vcvlvalaiv ylialavcqc
    121 rrknygqldi fpardtyhpm seyptyhthg ryvppsstdr spyekvsagn ggsslsytnp
    181 avaatsanl
    Mucin-1, isoform 20 precursor NP_001191225.1 (SEQ ID NO: 329)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 naipaptttk scretflkcf crfinkgvfw aspilssysd vpfpfsaqsg agvpgwgial
    121 lvlvcvlval aivylialav cqcrrknygq ldifpardty hpmseyptyh thgryvppss
    181 tdrspyekvs agnggsslsy tnpavaatsa nl
    Mucin-1, isoform 21 precursor NP_001191226.1 (SEQ ID NO: 330)
    1 mtpgtqspff llllltvlta ttapkpatvv tgsghasstp ggeketsatq rssvpsstek
    61 nalstgvsff flsfhisnlq fnssledpst dyyqelqrdi semavcqcrr knygqldifp
    121 ardtyhpmse yptyhthgry vppsstdrsp yekvsagngg sslsytnpav aatsanl
    N-myc proto-oncogene protein, isoform 1 NP_001280157.1, NP_005369.2 (SEQ ID
    NO: 331)
    1 mpscststmp gmicknpdle fdslqpcfyp deddfyfggp dstppgediw kkfellptpp
    61 lspsrgfaeh sseppswvte mllenelwgs paeedafglg glggltpnpv ilqdcmwsgf
    121 sareklerav seklqhgrgp ptagstaqsp gagaaspagr ghggaagagr agaalpaela
    181 hpaaecvdpa vvfpfpvnkr epapvpaapa sapaagpava sgagiaapag apgvapprpg
    241 grqtsggdhk alstsgedtl sdsddeddee edeeeeidvv tvekrrsssn tkavttftit
    301 vrpknaalgp graqsselil krclpihqqh nyaapspyve sedappqkki kseasprplk
    361 svippkaksl sprnsdseds errrnhnile rqrrndlrss fltlrdhvpe lvknekaakv
    421 vilkkateyv hslqaeehql llekeklqar qqqllkkieh artc
    N-myc proto-oncogene protein, isoform 2 NP_001280160.1 (SEQ ID NO: 332)
    1 mrgapgncvg aeqalarrkr aqtvairghp rppgppgdtr aesppdplqs agddeddeee
    61 deeeeidvvt vekrrsssnt kavttftitv rpknaalgpg raqsselilk rclpihqqhn
    121 yaapspyves edappqkkik seasprplks vippkaksls prnsdsedse rrrnhniler
    181 qrrndlrssf ltlrdhvpel vknekaakvv ilkkateyvh slqaeehqll lekeklqarq
    241 qqllkkieha rtc
    N-myc proto-oncogene protein, isoform 3 NP_001280162.1 (SEQ ID NO: 333)
    1 mrgapgncvg aeqalarrkr aqtvairghp rppgppgdtr aesppdplqs agvlevgagp
    61 rlprppregs tpgiktngae rspqspagrr adaellhvhh aghdlqeprp rv
    Cancer/testis antigen 1B NP_001318.1 (SEQ ID NO: 334)
    1 mqaegrgtgg stgdadgpgg pgipdgpggn aggpgeagat ggrgprgaga arasgpggga
    61 prgphggaas glngccrcga rgpesrllef ylampfatpm eaelarrsla qdapplpvpg
    121 vllkeftvsg niltirltaa dhrqlqlsis sclqqlsllm witqcflpvf laqppsgqrr
    Opioid growth factor receptor NP_031372.2 (SEQ ID NO: 335)
    1 mddpdcdstw eedeedaeda ededcedgea agardadagd edeeseepra arpssfqsrm
    61 tgsrnwratr dmcryrhnyp dlverdcngd tpnlsfyrne irflpngcfi edilqnwtdn
    121 ydllednhsy iqwlfplrep gvnwhakplt lrevevfkss qeiqerlvra yelmlgfygi
    181 rledrgtgtv graqnyqkrf qnlnwrshnn lritrilksl gelglehfqa plvrffleet
    241 lvrrelpgvr qsaldyfmfa vrcrhqrrql vhfawehfrp rckfvwgpqd klrrfkpssl
    301 phplegsrkv eeegspgdpd heastqgrtc gpehskgggr vdegpqprsv epqdagpler
    361 sqgdeagghg edrpeplspk eskkrklels rreqpptepg pqsaseveki alnlegcals
    421 qgslrtgtqe vggqdpgeav qpcrqplgar vadkvrkrrk vdegagdsaa vasggaqtla
    481 lagspapsgh pkaghsengv eedtegrtgp kegtpgspse tpgpspagpa gdepaespse
    541 tpgprpagpa gdepaespse tpgprpagpa gdepaespse tpgpspagpt rdepaespse
    601 tpgprpagpa gdepaespse tpgprpagpa gdepaespse tpgpspagpt rdepakagea
    661 aelqdaeves saksgkp
    P antigen family member 4 NP_001305806.1, NP_008934.1 (SEQ ID NO: 336)
    1 msarvrsrsr grgdgqeapd vvafvapges qqeepptdnq diepgqereg tppieerkve
    61 gdcqemdlek trsergdgsd vkektppnpk haktkeagdg qp
    Paired box protein Pax-3, isoform PAX3a NP_000429.2 (SEQ ID NO: 337)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkqvt tpdvekkiee
    121 ykrenpgmfs weirdkllkd avcdrntvps vssisrilrs kfgkgeeeea dlerkeaees
    181 ekkakhsidg ilsergkrwr lgrrtcwvtw rasas
    Paired box protein Pax-3, isoform PAX3i NP_001120838.1 (SEQ ID NO: 338)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkvtt pdvekkieey
    121 krenpgmfsw eirdkllkda vcdrntvpsv ssisrilrsk fgkgeeeead lerkeaeese
    181 kkakhsidgi lserasapqs degsdidsep dlplkrkqrr srttftaeql eelerafert
    241 hypdiytree laqrakltea rvqvwfsnrr arwrkqagan qlmafnhlip ggfpptampt
    301 lptyqlsets yqptsipqav sdpsstvhrp qplppstvhq stipsnpdss sayclpstrh
    361 gfssytdsfv ppsgpsnpmn ptignglspq vmglltnhgg vphqpqtdya lspltgglep
    421 tttvsascsq rldhmkslds lptsqsycpp tysttgysmd pvtgyqygqy gqsafhylkp
    481 dia
    Paired box protein Pax-3, isoform PAX3b NP_039230.1 (SEQ ID NO: 339)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkqvt tpdvekkiee
    121 ykrenpgmfs weirdkllkd avcdrntvps vssisrilrs kfgkgeeeea dlerkeaees
    181 ekkakhsidg ilsergkalv sgvssh
    Paired box protein Pax-3, isoform PAX3 NP_852122.1 (SEQ ID NO: 340)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkqvt tpdvekkiee
    121 ykrenpgmfs weirdkllkd avcdrntvps vssisrilrs kfgkgeeeea dlerkeaees
    181 ekkakhsidg ilserasapq sdegsdidse pdlplkrkqr rsrttftaeq leelerafer
    241 thypdiytre elaqraklte arvqvwfsnr rarwrkqaga nqlmafnhli pggfpptamp
    301 tlptyqlset syqptsipqa vsdpsstvhr pqplppstvh qstipsnpds ssayclpstr
    361 hgfssytdsf vppsgpsnpm nptignglsp qvmglltnhg gvphqpqtdy alspltggle
    421 ptttvsascs qrldhmksld slptsqsycp ptysttgysm dpvtgyqygq ygqskpwtf
    Paired box protein Pax-3, isoform PAX3d NP_852123.1 (SEQ ID NO: 341)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkqvt tpdvekkiee
    121 ykrenpgmfs weirdkllkd avcdrntvps vssisrilrs kfgkgeeeea dlerkeaees
    181 ekkakhsidg ilserasapq sdegsdidse pdlplkrkqr rsrttftaeq leelerafer
    241 thypdiytre elaqraklte arvqvwfsnr rarwrkqaga nqlmafnhli pggfpptamp
    301 tlptyqlset syqptsipqa vsdpsstvhr pqplppstvh qstipsnpds ssayclpstr
    361 hgfssytdsf vppsgpsnpm nptignglsp qvmglltnhg gvphqpqtdy alspltggle
    421 ptttvsascs qrldhmksld slptsqsycp ptysttgysm dpvtgyqygq ygqsafhylk
    481 pdia
    Paired box protein Pax-3, isoform PAX3e NP_852124.1 (SEQ ID NO: 342)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkqvt tpdvekkiee
    121 ykrenpgmfs weirdkllkd avcdrntvps vssisrilrs kfgkgeeeea dlerkeaees
    181 ekkakhsidg ilserasapq sdegsdidse pdlplkrkqr rsrttftaeq leelerafer
    241 thypdiytre elaqraklte arvqvwfsnr rarwrkqaga nqlmafnhli pggfpptamp
    301 tlptyqlset syqptsipqa vsdpsstvhr pqplppstvh qstipsnpds ssayclpstr
    361 hgfssytdsf vppsgpsnpm nptignglsp qvmglltnhg gvphqpqtdy alspltggle
    421 ptttvsascs qrldhmksld slptsqsycp ptysttgysm dpvtgyqygq ygqsafhylk
    481 pdiawfqill ntfdkssgee edleq
    Paired box protein Pax-3, isoform PAX3h NP_852125.1 (SEQ ID NO: 343)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkqvt tpdvekkiee
    121 ykrenpgmfs weirdkllkd avcdrntvps vssisrilrs kfgkgeeeea dlerkeaees
    181 ekkakhsidg ilserasapq sdegsdidse pdlplkrkqr rsrttftaeq leelerafer
    241 thypdiytre elaqraklte arvqvwfsnr rarwrkqaga nqlmafnhli pggfpptamp
    301 tlptyqlset syqptsipqa vsdpsstvhr pqplppstvh qstipsnpds ssayclpstr
    361 hgfssytdsf vppsgpsnpm nptignglsp qvpfiissqi slgfksf
    Paired box protein Pax-3, isoform PAX3g NP_852126.1 (SEQ ID NO: 344)
    1 mttlagavpr mmrpgpgqny prsgfplevs tplgqgrvnq lggvfingrp lpnhirhkiv
    61 emahhgirpc visrqlrvsh gcvskilcry qetgsirpga iggskpkqvt tpdvekkiee
    121 ykrenpgmfs weirdkllkd avcdrntvps vssisrilrs kfgkgeeeea dlerkeaees
    181 ekkakhsidg ilserasapq sdegsdidse pdlplkrkqr rsrttftaeq leelerafer
    241 thypdiytre elaqraklte arvqvwfsnr rarwrkqaga nqlmafnhli pggfpptamp
    301 tlptyqlset syqptsipqa vsdpsstvhr pqplppstvh qstipsnpds ssayclpstr
    361 hgfssytdsf vppsgpsnpm nptignglsp qvpfiissqi srk
    Paired box protein Pax-5, isoform 1 NP_057953.1 (SEQ ID NO: 345)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsivs tgsvtqvssv stdsagssys
    181 isgilgitsp sadtnkrkrd egiqespvpn ghslpgrdfl rkqmrgdlft qqqlevldrv
    241 ferqhysdif tttepikpeq tteysamasl agglddmkan lasptpadig ssvpgpqsyp
    301 ivtgrdlast tlpgypphvp pagqgsysap tltgmvpgse fsgspyshpq yssyndswrf
    361 pnpgllgspy yysaaargaa ppaaataydr h
    Paired box protein Pax-5, isoform 2 NP_001267476.1 (SEQ ID NO: 346)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsivs tgsvtqvssv stdsagssys
    181 isgilgitsp sadtnkrkrd egiqespvpn ghslpgrdfl rkqmrgdlft qqqlevldrv
    241 ferqhysdif tttepikpeq tteysamasl agglddmkan lasptpadig ssvpgpqsyp
    301 ivtgsefsgs pyshpqyssy ndswrfpnpg llgspyyysa aargaappaa ataydrh
    Paired box protein Pax-5, isoform 3 NP_001267477.1 (SEQ ID NO: 347)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsivs tgsvtqvssv stdsagssys
    181 isgilgitsp sadtnkrkrd egiqespvpn ghslpgrdfl rkqmrgdlft qqqlevldrv
    241 ferqhysdif tttepikpeq tteysamasl agglddmkan lasptpadig ssvpgpqsyp
    301 ivtgrdlast tlpgypphvp pagqgsysap tltgmvpgsp yyysaaarga appaaatayd
    361 rh
    Paired box protein Pax-5, isoform 4 NP_001267478.1 (SEQ ID NO: 348)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsivs tgsvtqvssv stdsagssys
    181 isgilgitsp sadtnkrkrd egiqespvpn ghslpgrdfl rkqmrgdlft qqqlevldrv
    241 ferqhysdif tttepikpeq gvsfpgvpta tlsiprtttp ggsptrgcla pptiialppe
    301 epphlqpplp mtvtdpwsqa gtkh
    Paired box protein Pax-5, isoform 5 NP_001267479.1 (SEQ ID NO: 349)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsivs tgsvtqvssv stdsagssys
    181 isgilgitsp sadtnkrkrd egiqespvpn ghslpgrdfl rkqmrgdlft qqqlevldrv
    241 ferqhysdif tttepikpeq apptiialpp eepphlqppl pmtvtdpwsq agtkh
    Paired box protein Pax-5, isoform 6 NP_001267480.1 (SEQ ID NO: 350)
    1 mfaweirdrl laervcdndt vpsyssinri irtkvqqppn qpvpasshsi vstgsvtqvs
    61 systdsagss ysisgilgit spsadtnkrk rdegiqespv pnghslpgrd flrkqmrgdl
    121 ftqqqlevld rvferqhysd iftttepikp eqtteysama slagglddmk anlasptpad
    181 igssvpgpqs ypivtgspyy ysaaargaap paaataydrh
    Paired box protein Pax-5, isoform 7 NP_001267481.1 (SEQ ID NO: 351)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsivs tgsvtqvssv stdsagssys
    181 isgilgitsp sadtnkrkrd egiqespvpn ghslpgrdfl rkqmrgdlft qqqlevldrv
    241 ferqhysdif tttepikpeq tteysamasl agglddmkan lasptpadig ssvpgpqsyp
    301 ivtgspyyys aaargaappa aataydrh
    Paired box protein Pax-5, isoform 8 NP_001267482.1 (SEQ ID NO: 352)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsigi qespvpnghs lpgrdflrkg
    181 mrgdlftqqq levldrvfer qhysdifttt epikpeqtte ysamaslagg lddmkanlas
    241 ptpadigssv pgpqsypivt grdlasttlp gypphvppag qgsysaptlt gmvpgspyyy
    301 saaargaapp aaataydrh
    Paired box protein Pax-5, isoform 9 NP_001267483.1 (SEQ ID NO: 353)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg ryyetgsikp gviggskpkv atpkvvekia eykrqnptmf aweirdrlla
    121 ervcdndtvp svssinriir tkvqqppnqp vpasshsigi qespvpnghs lpgrdflrkg
    181 mrgdlftqqq levldrvfer qhysdifttt epikpeqtte ysamaslagg lddmkanlas
    241 ptpadigssv pgpqsypivt grdlasttlp gypphvppag qgsysaptlt gmvpgsefsg
    301 spyshpqyss yndswrfpnp gllgspyyys aaargaappa aataydrh
    Paired box protein Pax-5, isoform 10 NP_001267484.1 (SEQ ID NO: 354)
    1 mdleknyptp rtsrtghggv nqlggvfvng rplpdvvrqr ivelahqgvr pcdisrqlrv
    61 shgcvskilg riirtkvqqp pnqpvpassh sivstgsvtq vssvstdsag ssysisgilg
    121 itspsadtnk rkrdegiqes pvpnghslpg rdflrkqmrg dlftqqqlev ldrvferqhy
    181 sdiftttepi kpeqtteysa maslaggldd mkanlasptp adigssvpgp qsypivtgse
    241 fsgspyshpq yssyndswrf pnpgllgspy yysaaargaa ppaaataydr h
    Paired box protein Pax-5, isoform 11 NP_001267485.1 (SEQ ID NO: 355)
    1 mfaweirdrl laervcdndt vpsvssinri irtkvqqppn qpvpasshsi vstgsvtqvs
    61 systdsagss ysisgilgit spsadtnkrk rdegiqespv pnghslpgrd flrkqmrgdl
    121 ftqqqlevld rvferqhysd iftttepikp eqtteysama slagglddmk anlasptpad
    181 igssvpgpqs ypivtgrdla sttlpgypph vppagqgsys aptltgmvpg sefsgspysh
    241 pqyssyndsw rfpnpgllgs pyyysaaarg aappaaatay drh
    Platelet-derived growth factor receptor beta, isoform 1 NP_002600.1 (SEQ ID
    NO: 356)
    1 mrlpgampal alkgelllls lllllepqis qglvvtppgp elvlnvsstf vltcsgsapv
    61 vwermsqepp qemakaqdgt fssvltltnl tgldtgeyfc thndsrglet derkrlyifv
    121 pdptvgflpn daeelfiflt eiteitiper vtdpqlvvtl hekkgdvalp vpydhqrgfs
    181 gifedrsyic kttigdrevd sdayyvyrlq vssinvsvna vqtvvrqgen itlmcivign
    241 evvnfewtyp rkesgrlvep vtdflldmpy hirsilhips aeledsgtyt cnvtesvndh
    301 qdekainitv vesgyvrllg evgtlqfael hrsrtlqvvf eayppptvlw fkdnrtlgds
    361 sageialstr nvsetryvse ltivrvkvae aghytmrafh edaevqlsfq lqinvpvrvl
    421 elseshpdsg eqtvrcrgrg mpqpniiwsa crdlkrcpre lpptllgnss eeesqletnv
    481 tyweeeqefe vvstlrlqhv drplsvrctl rnavgqdtqe vivvphslpf kvvvisaila
    541 lvvltiisli ilimlwqkkp ryeirwkvie svssdgheyi yvdpmqlpyd stwelprdql
    601 vlgrtlgsga fgqvveatah glshsqatmk vavkmlksta rssekqalms elkimshlgp
    661 hlnvvnllga ctkggpiyii teycrygdlv dylhrnkhtf lqhhsdkrrp psaelysnal
    721 pvglplpshv sltgesdggy mdmskdesvd yvpmldmkgd vkyadiessn ymapydnyvp
    781 sapertcrat linespvlsy mdlvgfsyqv angmeflask ncvhrdlaar nvlicegklv
    841 kicdfglard imrdsnyisk gstflplkwm apesifnsly ttlsdvwsfg illweiftlg
    901 gtpypelpmn eqfynaikrg yrmaqpahas deiyeimqkc weekfeirpp fsqlvlller
    961 llgegykkky qqvdeeflrs dhpailrsqa rlpgfhglrs pldtssvlyt avqpnegdnd
    1021 yiiplpdpkp evadegpleg spslasstln evntsstisc dsplepqdep epepqlelqv
    1081 epepeleqlp dsgcpaprae aedsfl
    Platelet-derived growth factor receptor beta, isoform 2 NP_001341945.1 (SEQ
    ID NO: 357)
    1 msqeppqema kaqdgtfssv ltltnltgld tgeyfcthnd srgletderk rlyifvpdpt
    61 vgflpndaee lfiflteite itipervtdp qlvvtlhekk gdvalpvpyd hqrgfsgife
    121 drsyicktti gdrevdsday yvyrlqvssi nvsvnavqtv vrqgenitlm civignevvn
    181 fewtyprkes grlvepvtdf lldmpyhirs ilhipsaele dsgtytcnvt esvndhqdek
    241 ainitvvesg yvrllgevgt lqfaelhrsr tlqvvfeayp pptvlwfkdn rtlgdssage
    301 ialstrnvse tryvseltlv rvkvaeaghy tmrafhedae vqlsfqlqin vpvrvlelse
    361 shpdsgeqtv rcrgrgmpqp niiwsacrdl krcprelppt llgnsseees qletnvtywe
    421 eeqefevvst lrlqhvdrpl svrctlrnav gqdtqevivv phslpfkvvv isailalvvl
    481 tiisliilim lwqkkpryei rwkviesyss dgheyiyvdp mqlpydstwe lprdqlvlgr
    541 tlgsgafgqv veatahglsh sqatmkvavk mlkstarsse kqalmselki mshlgphlnv
    601 vnllgactkg gpiyiiteyc rygdlvdylh rnkhtflqhh sdkrrppsae lysnalpvgl
    661 plpshvsltg esdggymdms kdesvdyvpm ldmkgdvkya diessnymap ydnyvpsape
    721 rtcratline spvlsymdlv gfsyqvangm eflaskncvh rdlaarnvli cegklvkicd
    781 fglardimrd snyiskgstf lplkwmapes ifnslyttls dvwsfgillw eiftlggtpy
    841 pelpmneqfy naikrgyrma qpahasdeiy eimqkcweek feirppfsql vlllerllge
    901 gykkkyqqvd eeflrsdhpa ilrsqarlpg fhglrspldt ssvlytavqp negdndyiip
    961 lpdpkpevad egplegspsl asstlnevnt sstiscdspl epqdepepep qlelqvepep
    1021 eleqlpdsgc papraeaeds fl
    Platelet-derived growth factor receptor beta, isoform 3 NP_001341946.1 (SEQ
    ID NO: 358)
    1 mitnvaflvs lrteatsakp plgtgrwilm ptmstdsrvs plsglmlsry ssinvsvnav
    61 qtvvrqgeni tlmcivigne vvnfewtypr kesgrlvepv tdflldmpyh irsilhipsa
    121 eledsgtytc nvtesvndhq dekainitvv esgyvrllge vgtlqfaelh rsrtlqvvfe
    181 ayppptvlwf kdnrtlgdss ageialstrn vsetryvsel tivrvkvaea ghytmrafhe
    241 daevqlsfql qinvpvrvle lseshpdsge qtvrcrgrgm pqpniiwsac rdlkrcprel
    301 pptllgnsse eesqletnvt yweeeqefev vstlrlqhvd rplsvrctlr navgqdtgev
    361 ivvphslpfk vvvisailal vvltiislii limlwqkkpr yeirwkvies vssdgheyiy
    421 vdpmqlpyds twelprdqlv lgrtlgsgaf gqvveatahg lshsqatmkv avkmlkstar
    481 ssekqalmse lkimshlgph lnvvnllgac tkggpiyiit eycrygdlvd ylhrnkhtfl
    541 qhhsdkrrpp saelysnalp vglplpshvs ltgesdggym dmskdesvdy vpmldmkgdv
    601 kyadiessny mapydnyvps apertcratl inespvlsym dlvgfsyqva ngmeflaskn
    661 cvhrdlaarn vlicegklvk icdfglardi mrdsnyiskg stflplkwma pesifnslyt
    721 tlsdvwsfgi llweiftlgg tpypelpmne qfynaikrgy rmaqpahasd eiyeimqkcw
    781 eekfeirppf sqlvlllerl lgegykkkyq qvdeeflrsd hpailrsqar lpgfhglrsp
    841 ldtssvlyta vqpnegdndy iiplpdpkpe vadegplegs pslasstlne vntsstiscd
    901 splepqdepe pepqlelqve pepeleqlpd sgcpapraea edsfl
    Placenta-specific protein 1 precursor NP_001303816.1, NP_001303817.1,
    NP_001303818.1, NP_068568.1 (SEQ ID NO: 359)
    1 mkvfkfiglm illtsafsag sgqspmtvlc sidwfmvtvh pfmlnndvcv hfhelhlglg
    61 cppnhvqpha yqftyrvtec girakaysqd mviysteihy sskgtpskfv ipvscaapqk
    121 spwltkpcsm rvasksrata qkdekcyevf slsqssqrpn cdcppcvfse eehtqvpchq
    181 agaqeaqplq pshfldised wslhtddmig sm
    Melanoma antigen preferentially expressed in tumors, isoform a
    NP_001278644.1, NP_001278645.1, NP_006106.1, NP_996836.1, NP_996837.1,
    NP_996838.1, NP_996839.1 (SEQ ID NO: 360)
    1 merrrlwgsi qsryismsvw tsprrlvela gqsllkdeal aiaalellpr elfpplfmaa
    61 fdgrhsqtlk amvqawpftc lplgvlmkgq hlhletfkav ldgldvllaq evrprrwklq
    121 vldlrknshq dfwtvwsgnr aslysfpepe aaqpmtkkrk vdglsteaeq pfipvevlvd
    181 lflkegacde lfsyliekvk rkknvlrlcc kklkifampm qdikmilkmv qldsiedlev
    241 tctwklptla kfspylgqmi nlrrlllshi hassyispek eeqyiaqfts qflslqclqa
    301 lyvdslfflr grldqllrhv mnpletlsit ncrlsegdvm hlsqspsysq lsvlslsgvm
    361 ltdvspeplq allerasatl qdlvfdecgi tddqllallp slshcsqltt lsfygnsisi
    421 salqsllqhl iglsnlthvl ypvplesyed ihgtlhlerl aylharlrel lcelgrpsmv
    481 wlsanpcphc gdrtfydpep ilcpcfmpn
    Melanoma antigen preferentially expressed in tumors, isoform b
    NP_001278646.1, NP_001278648.1, NP_001305055.1, NP_001305056.1 (SEQ ID NO:
    361)
    1 msvwtsprrl velagqsllk dealaiaale llprelfppl fmaafdgrhs qtlkamvqaw
    61 pftclplgvl mkgqhlhlet fkavldgldv llaqevrprr wklqvldlrk nshqdfwtvw
    121 sgnraslysf pepeaaqpmt kkrkvdglst eaeqpfipve vlvdlflkeg acdelfsyli
    181 ekvkrkknvl rlcckklkif ampmqdikmi lkmvqldsie dlevtctwkl ptlakfspyl
    241 gqminlrrll lshihassyi spekeeqyia qftsqflslq clqalyvdsl fflrgrldql
    301 lrhvmnplet lsitncrlse gdvmhlsqsp sysqlsvlsl sgvmltdvsp eplqallera
    361 satlqdlvfd ecgitddqll allpslshcs qlttlsfygn sisisalqsl lqhliglsnl
    421 thvlypvple syedihgtlh lerlaylhar lrellcelgr psmvwlsanp cphcgdrtfy
    481 dpepilcpcf mpn
    Phosphatidylinositol
     3,4,5-triphosphate-dependent Rac exchanger 2 protein,
    isoform a NP_079146.2 (SEQ ID NO: 362)
    1 msedsrgdsr aesakdlekq lrlrvcvlse lqkterdyvg tleflvsafl hrmnqcaask
    61 vdknvteetv kmlfsniedi lavhkeflkv veeclhpepn aqqevgtcfl hfkdkfriyd
    121 eycsnhekaq klllelnkir tirtfllncm llggrkntdv plegylvtpi qrickyplil
    181 kellkrtprk hsdyaavmea lqamkavcsn ineakrqmek levleewqsh iegwegsnit
    241 dtctemlmcg vllkissgni qervfflfdn llvyckrkhr rlknskastd ghrylfrgri
    301 ntevmevenv ddgtadfhss ghivvngwki hntaknkwfv cmaktpeekh ewfeailker
    361 errkglklgm eqdtwvmise qgeklykmmc rqgnlikdrk rklttfpkcf lgsefvswll
    421 eigeihrpee gvhlgqalle ngiihhvtdk hqfkpeqmly rfryddgtfy prnemqdvis
    481 kgvrlycrlh slftpvirdk dyhlrtyksv vmanklidwl iaqgdcrtre eamifgvglc
    541 dngfmhhvle ksefkdepll frffsdeeme gsnmkhrlmk hdlkvvenvi akslliksne
    601 gsygfgledk nkvpiiklve kgsnaemagm evgkkifain gdlvfmrpfn evdcflkscl
    661 nsrkplrvlv stkpretvki pdsadglgfq irgfgpsvvh avgrgtvaaa aglhpgqcii
    721 kvnginvske thasviahvt acrkyrrptk qdsiqwvyns iesaqedlqk shskppgdea
    781 gdafdckvee vidkfntmai idgkkehvsl tvdnvhleyg vvyeydstag ikcnvvekmi
    841 epkgffslta kilealaksd ehfvqnctsl nslneviptd lqskfsalcs eriehlcqri
    901 ssykkfsrvl knrawptfkq akskisplhs sdfcptnchv nvmevsypkt stslgsafgv
    961 qldsrkhnsh dkenksseqg klspmvyiqh tittmaapsg lslgqqdghg lryllkeedl
    1021 etqdiyqkll gklqtalkev emcvcqiddl lssityspkl erktsegiip tdsdnekger
    1081 nskrvcfnva gdeqedsghd tisnrdsysd cnsnrnsias ftsicssqcs syfhsdemds
    1141 gdelplsvri shdkqdkihs clehlfsqvd sitnllkgqa vvrafdqtky ltpgrglqef
    1201 qqemepklsc pkrlrlhikq dpwnlpssvr tlaqnirkfv eevkcrllla lleysdsetq
    1261 lrrdmvfcqt lvatvcafse qlmaalnqmf dnskenemet weasrrwldq ianagvlfhf
    1321 qsllspnitd eqamledtiv alfdlekvsf yfkpseeepl vanvpltyqa egsrqalkvy
    1381 fyidsyhfeq lpqrlknggg fkihpvlfaq alesmegyyy rdnvsveefq aqinaaslek
    1441 vkqynqklra fyldksnspp nstskaayvd klmrpinald elyrlvasfi rskrtaacan
    1501 tacsasgvgl lsysselcnr lgachiimcs sgvhrctlsv tleqaiilar shglppryim
    1561 qatdvmrkqg arvqntaknl gvrdrtpqsa prlyklcepp ppagee
    Phosphatidylinositol  3,4,5-triphosphate-dependent Rac exchanger 2 protein,
    isoform b NP_079446.3 (SEQ ID NO: 363)
    1 msedsrgdsr aesakdlekq lrlrvcvlse lqkterdyvg tleflvsafl hrmnqcaask
    61 vdknvteetv kmlfsniedi lavhkeflkv veeclhpepn aqqevgtcfl hfkdkfriyd
    121 eycsnhekaq klllelnkir tirtfllncm llggrkntdv plegylvtpi qrickyplil
    181 kellkrtprk hsdyaavmea lqamkavcsn ineakrqmek levleewqsh iegwegsnit
    241 dtctemlmcg vllkissgni qervfflfdn llvyckrkhr rlknskastd ghrylfrgri
    301 ntevmevenv ddgtadfhss ghivvngwki hntaknkwfv cmaktpeekh ewfeailker
    361 errkglklgm eqdtwvmise qgeklykmmc rqgnlikdrk rklttfpkcf lgsefvswll
    421 eigeihrpee gvhlgqalle ngiihhvtdk hqfkpeqmly rfryddgtfy prnemqdvis
    481 kgvrlycrlh slftpvirdk dyhlrtyksv vmanklidwl iaqgdcrtre eamifgvglc
    541 dngfmhhvle ksefkdepll frffsdeeme gsnmkhrlmk hdlkvvenvi akslliksne
    601 gsygfgledk nkvpiiklve kgsnaemagm evgkkifain gdlvfmrpfn evdcflkscl
    661 nsrkplrvlv stkpretvki pdsadglgfq irgfgpsvvh avgrgtvaaa aglhpgqcii
    721 kvnginvske thasviahvt acrkyrrptk qdsiqwvyns iesaqedlqk shskppgdea
    781 gdafdckvee vidkfntmai idgkkehvsl tvdnvhleyg vvyeydstag ikcnvvekmi
    841 epkgffslta kilealaksd ehfvqnctsl nslneviptd lqskfsalcs eriehlcqri
    901 ssykkvqase rfynftarha vwehsfdlhs vsstfpvpvt meflllpppl lgisqdgrqh
    961 cipedlpsqe mllaerapv
    Protamine-2, isoform 1 NP_002753.2 (SEQ ID NO: 364)
    1 mvryrvrsls ershevyrqq lhgqeqghhg qeeqglspeh vevyerthgq shyrrrhcsr
    61 rrlhrihrrq hrscrrrkrr scrhrrrhrr gcrtrkrtcr rh
    Protamine-2, isoform 2 NP_001273285.1 (SEQ ID NO: 365)
    1 mvryrvrsls ershevyrqq lhgqeqghhg qeeqglspeh vevyerthgq shyrrrhcsr
    61 rrlhrihrrq hrscrrrkrr scrhrrrhrr eslgdplnqn flsqkaaepg rehaegtklp
    121 gpltpswklr ksrpkhqvrp
    Protamine-2, isoform 3 NP_001273286.1 (SEQ ID NO: 366)
    1 mvryrvrsls ershevyrqq lhgqeqghhg qeeqglspeh vevyerthgq shyrrrhcsr
    61 rrlhrihrrq hrscrrh
    Protamine-2, isoform 4 NP_001273287.1 (SEQ ID NO: 367)
    1 mvryrvrsls ershevyrqq lhgqeqghhg qeeqglspeh vevyerthgq shyrrrhcsr
    61 rrlhrihrrq hrscrrrkrr scrhrrrhrr epgrehaegt klpgpltpsw klrksrpkhq
    121 vrp
    Protamine-2, isoform 5 NP_001273288.1 (SEQ ID NO: 368)
    1 mvryrvrsls ershevyrqq lhgqeqghhg qeeqglspeh vevyerthgq shyrrrhcsr
    61 rrlhrihrrq hrscrrrkrr scrhrrrhrr glpapppcpa cp
    Progranulin NP_002078.1 (SEQ ID NO: 369)
    1 mwtlvswval taglvagtrc pdgqfcpvac cldpggasys ccrplldkwp ttlsrhlggp
    61 cqvdahcsag hsciftvsgt ssccpfpeav acgdghhccp rgfhcsadgr scfqrsgnns
    121 vgaiqcpdsq fecpdfstcc vmvdgswgcc pmpqascced rvhccphgaf cdlvhtrcit
    181 ptgthplakk lpaqrtnrav alsssvmcpd arsrcpdgst ccelpsgkyg ccpmpnatcc
    241 sdhlhccpqd tvcdliqskc lskenattdl ltklpahtvg dvkcdmevsc pdgytccrlq
    301 sgawgccpft qavccedhih ccpagftcdt qkgtceqgph qvpwmekapa hlslpdpqal
    361 krdvpcdnvs scpssdtccq ltsgewgccp ipeavccsdh qhccpqgytc vaegqcqrgs
    421 eivaglekmp arraslshpr digcdqhtsc pvgqtccpsl ggswaccqlp havccedrqh
    481 ccpagytcnv karscekevv saqpatflar sphvgvkdve cgeghfchdn qtccrdnrqg
    541 waccpyrqgv ccadrrhccp agfrcaargt kclrreaprw daplrdpalr qll
    Myeloblastin precursor NP_002768.3 (SEQ ID NO: 370)
    1 mahrppspal asvllallls gaaraaeivg gheaqphsrp ymaslqmrgn pgshfcggtl
    61 ihpsfvltaa hclrdipqrl vnvvlgahnv rtqeptqqhf svaqvflnny daenklndvl
    121 liqlsspanl sasvatvqlp qqdqpvphgt qclamgwgrv gandppaqvl qelnvtvvtf
    181 fcrphnictf vprrkagicf gdsggplicd giiqgidsfv iwgcatrlfp dfftrvalyv
    241 dwirstlrrv eakgrp
    Prostate stem cell antigen preportein NP_005663.2 (SEQ ID NO: 371)
    1 maglalqpgt allcysckaq vsnedclqve nctqlgeqcw tariravgll tviskgcsln
    61 cvddsqdyyv gkknitccdt dlcnasgaha lqpaaailal lpalglllwg pgql
    Ras-related C3 botulinum toxin substrate 1 isoform Raclb NP_061485.1 (SEQ ID
    NO: 372)
    1 mqaikcvvvg dgavgktcll isyttnafpg eyiptvfdny sanvmvdgkp vnlglwdtag
    61 qedydrlrpl sypqtvgety gkditsrgkd kpiadvflic fslvspasfe nvrakwypev
    121 rhhcpntpii lvgtkldlrd dkdtieklke kkltpitypq glamakeiga vkylecsalt
    181 qrglktvfde airavlcppp vkkrkrkcll l
    Regenerating islet-derived protein 3-alpha precursor NP_002571.1,
    NP_620354.1, NP_620355.1 (SEQ ID NO: 373)
    1 mlppmalpsv swmllsclml lsqvqgeepq relpsarirc pkgskaygsh cyalflspks
    61 wtdadlacqk rpsgnlvsvl sgaegsfvss lvksignsys yvwiglhdpt qgtepngegw
    121 ewsssdvmny fawernpsti sspghcasls rstaflrwkd yncnvrlpyv ckftd
    Regulator of G-protein signaling 5, isoform 1 NP_003608.1 (SEQ ID NO: 374)
    1 mckglaalph sclerakeik iklgillqkp dsvgdlvipy nekpekpakt qktsldealq
    61 wrdsldkllq nnyglasfks flksefseen lefwiacedy kkikspakma ekakqiyeef
    121 iqteapkevn idhftkditm knlvepslss fdmaqkriha lmekdslprf vrsefyqeli
    181 k
    Regulator of G-protein signaling 5, isoform 2 NP_001182232.1, NP_001241677.1
    (SEQ ID NO: 375)
    1 maekakqiye efiqteapke vnidhftkdi tmknlvepsl ssfdmaqkri halmekdslp
    61 rfvrsefyqe lik
    Regulator of G-protein signaling 5, isoform 3 NP_001241678.1 (SEQ ID NO: 376)
    1 mckglaalph sclerakeik iklgillqkp dsvgdlvipy nekpekpakt qktsldealq
    61 wrdsldkllq nnyglasfks flksefseen lefwiacedy kkikspakma ekakqiyeef
    121 iqteapkevg lwvnidhftk ditmknlvep slssfdmaqk rihalmekds lprfvrsefy
    181 qelik
    Rho-related GTP-binding protein RhoC precursor NP_001036143.1,
    NP_001036144.1, NP_786886.1 (SEQ ID NO: 377)
    1 maairkklvi vgdgacgktc llivfskdqf pevyvptvfe nyiadievdg kqvelalwdt
    61 agqedydrlr plsypdtdvi lmcfsidspd slenipekwt pevkhfcpnv piilvgnkkd
    121 lrqdehtrre lakmkqepvr seegrdmanr isafgylecs aktkegvrev fematraglq
    181 vrknkrrrgc pil
    Sarcoma antigen 1 NP_061136.2 (SEQ ID NO: 378)
    1 mqasplqtsq ptppeelhaa ayvftndgqq mrsdevnlva tghqskkkhs rkskrhsssk
    61 rrksmsswld kqedaavths iceerinngq pvadnvlsta ppwpdatiah nireermeng
    121 qsrtdkvlst appqlvhmaa agipsmstrd lhstvthnir eermengqpq pdnvlstgpt
    181 glinmaatpi pamsardlya tvthnvceqk menvqpapdn vlltlrprri nmtdtgispm
    241 strdpyatit ynvpeekmek gqpqpdnils tastglinva gagtpaistn glystvphnv
    301 ceekmendqp qpnnvlstvq pviiyltatg ipgmntrdqy atithnvcee rvvnnqplps
    361 nalstvlpgl aylatadmpa mstrdqhati ihnlreekkd nsqptpdnvl savtpelinl
    421 agagippmst rdqyatvnhh vhearmengq rkqdnvlsnv lsglinmaga sipamssrdl
    481 yatithsvre ekmesgkpqt dkvisndapq lghmaaggip smstkdlyat vtqnvheerm
    541 ennqpqpsyd lstvlpglty ltvagipams trdqyatvth nvheekikng qaasdnvfst
    601 vppafinmaa tgvssmstrd qyaavthnir eekinnsqpa pgnilstapp wlrhmaaagi
    661 sstitrdlyv tathsvheek mtngqqapdn slstvppgci nlsgagiscr strdlyatvi
    721 hdiqeeemen dqtppdgfls nsdspelinm tghcmppnal dsfshdftsl skdellykpd
    781 snefavgtkn ysysagdppv tvmslvetvp ntpqispama kkinddikyq lmkevrrfgq
    841 nyerifille evqgsmkvkr qfveftikea arfkkvvliq qlekalkeid shchlrkvkh
    901 mrkr
    Squamous cell carcinoma antigen recognized by T-cells 3 NP_055521.1 (SEQ ID
    NO: 379)
    1 mataaetsas epeaeskagp kadgeedevk aartrrkvls ravaaatykt mgpawdqqee
    61 gvsesdgdey amassaessp geyeweydee eeknqleier leeqlsinvy dynchvdlir
    121 llrlegeltk vrmarqkmse ifplteelwl ewlhdeisma qdgldrehvy dlfekavkdy
    181 icpniwleyg qysvggigqk gglekvrsvf eralssvglh mtkglalwea yrefesaive
    241 aarlekvhsl frrqlaiply dmeatfaeye ewsedpipes viqnynkalq qlekykpyee
    301 allqaeaprl aeyqayidfe mkigdpariq liferalven clvpdlwiry sqyldrqlkv
    361 kdlvlsvhnr airncpwtva lwsryllame rhgvdhqvis vtfekalnag fiqatdyvei
    421 wqayldylrr rvdfkqdssk eleelraaft raleylkqev eerfnesgdp scvimqnwar
    481 iearlcnnmq karelwdsim trgnakyanm wleyynlera hgdtqhcrka lhravqctsd
    541 ypehvcevll tmertegsle dwdiavqkte trlarvneqr mkaaekeaal vqqeeekaeq
    601 rkraraekka lkkkkkirgp ekrgadedde kewgddeeeq pskrrrvens ipaagetqnv
    661 evaagpagkc aavdveppsk gkekaaslkr dmpkvlhdss kdsitvfvsn lpysmqepdt
    721 klrplfeacg evvqirpifs nrgdfrgycy vefkeeksal qalemdrksv egrpmfvspc
    781 vdksknpdfk vfrystslek hklfisglpf sctkeeleei ckahgtvkdl rlvtnragkp
    841 kglayveyen esqasqavmk mdgmtikeni ikvaisnppq rkvpekpetr kapggpmllp
    901 qtygargkgr tqlsllpral qrpsaaapqa engpaaapav aapaateapk msnadfaklf
    961 lrk
    Secretory leukocyte protein inhibitor NP_003055.1 (SEQ ID NO: 380)
    1 mkssglfpfl vllalgtlap wavegsgksf kagvcppkks aqclrykkpe cqsdwqcpgk
    61 krccpdtcgi kcldpvdtpn ptrrkpgkcp vtyggclmln ppnfcemdgq ckrdlkccmg
    121 mcgkscvspv ka
    Transcription factor SOX-10 NP_008872.1 (SEQ ID NO: 381)
    1 maeeqdlsev elspvgseep rclspgsaps lgpdgggggs glraspgpge lgkvkkeqqd
    61 geadddkfpv cireaysqvl sgydwtivpm pvrvngasks kphvkrpmna fmvwaqaarr
    121 kladqyphlh naelsktlgk lwrllnesdk rpfieeaerl rmqhkkdhpd ykyqprrrkn
    181 gkaaqgeaec pggeaeqggt aaiqahyksa hldhrhpgeg spmsdgnpeh psgqshgppt
    241 ppttpktelq sgkadpkrdg rsmgeggkph idfgnvdige ishevmsnme tfdvaeldqy
    301 lppnghpghv ssysaagygl gsalavasgh sawiskppgv alptvsppgv dakaqvktet
    361 agpqgpphyt dqpstsqiay tslslphygs afpsisrpqf dysdhqpsgp yyghsgqasg
    421 lysafsymgp sqrplytais dpspsgpqsh spthweqpvy ttlsrp
    Sperm surface protein Sp17 NP_059121.1 (SEQ ID NO: 382)
    1 msipfsnthy ripqgfgnll egltreilre qpdnipafaa ayfesllekr ektnfdpaew
    61 gskvedrfyn nhafeeqepp eksdpkqees qisgkeeets vtildsseed kekeevaavk
    121 iqaafrghia reeakkmktn slqneekeen k
    Protein SSX2, isoform a NP_003138.3 (SEQ ID NO: 383)
    1 mngddafarr ptvgaqipek iqkafddiak yfskeewekm kasekifyvy mkrkyeamtk
    61 lgfkatlppf mcnkraedfq gndldndpnr gnqverpqmt fgrlqgispk impkkpaeeg
    121 ndseevpeas gpqndgkelc ppgkpttsek ihersgnrea qekeerrgta hrwssqnthn
    181 igrfslstsm gavhgtpkti thnrdpkggn mpgptdcvre nsw
    Protein SSX2, isoform b NP_783629.1 (SEQ ID NO: 384)
    1 mngddafarr ptvgaqipek iqkafddiak yfskeewekm kasekifyvy mkrkyeamtk
    61 lgfkatlppf mcnkraedfq gndldndpnr gnqverpqmt fgrlqgispk impkkpaeeg
    121 ndseevpeas gpqndgkelc ppgkpttsek ihersgpkrg ehawthrlre rkqlviyeei
    181 sdpeedde
    Protein SSX2, isoform c NP_001265626.1 (SEQ ID NO: 385)
    1 mngddafarr ptvgaqipek iqkafddiak yfskeewekm kasekifyvy mkrkyeamtk
    61 lgfkatlppf mcnkraedfq gndldndpnr gnqverpqmt fgrlqgispk impkkpaeeg
    121 ndseevpeas gpqndgkelc ppgkpttsek ihersgnrea qekeerrgta hrwssqnthn
    181 igpkrgehaw thrlrerkql viyeeisdpe edde
    Lactosylceramide alpha-2,3-sialyltransferase, isoform 1 NP_003887.3 (SEQ ID
    NO: 386)
    1 mrtkaagcae rrplqprtea aaapagramp seytyvklrs dcsrpslqwy traqskmrrp
    61 slllkdilkc tllvfgvwil yilklnytte ecdmkkmhyv dpdhvkraqk yaqqvlqkec
    121 rpkfaktsma llfehrysvd llpfvqkapk dseaeskydp pfgfrkfssk vqtllellpe
    181 hdlpehlkak tcrrcvvigs ggilhglelg htlnqfdvvi rlnsapvegy sehvgnktti
    241 rmtypegapl sdleyysndl fvavlfksvd fnwlqamvkk etlpfwvrlf fwkqvaekip
    301 lqpkhfriln pviiketafd ilqysepqsr fwgrdknvpt igviavvlat hlcdevslag
    361 fgydlnqprt plhyfdsqcm aamnfqtmhn vttetkfllk lvkegvvkdl sggidref
    Lactosylceramide alpha-2,3-sialyltransferase, isoform 2 NP_001035902.1 (SEQ
    ID NO: 387)
    1 masvpmpsey tyvklrsdcs rpslqwytra qskmrrpsll lkdilkctll vfgvwilyil
    61 klnytteecd mkkmhyvdpd hvkraqkyaq qvlqkecrpk faktsmallf ehrysvdllp
    121 fvqkapkdse aeskydppfg frkfsskvqt llellpehdl pehlkaktcr rcvvigsggi
    181 lhglelghtl nqfdvvirin sapvegyseh vgnkttirmt ypegaplsdl eyysndlfva
    241 vlfksvdfnw lqamvkketl pfwvrlffwk qvaekiplqp khfrilnpvi iketafdilq
    301 ysepqsrfwg rdknvptigv iavvlathlc devslagfgy dlnqprtplh yfdsqcmaam
    361 nfqtmhnvtt etkfllklvk egvvkdlsgg idref
    Lactosylceramide alpha-2,3-sialyltransferase, isoform 3 NP_001341152.1,
    NP_001341153.1, NP_001341155.1, NP_001341162.1, NP_001341163.1,
    NP_001341177.1 (SEQ ID NO: 388)
    1 mallfehrys vdllpfvqka pkdseaesky dppfgfrkfs skvqtllell pehdlpehlk
    61 aktcrrcvvi gsggilhgle lghtlnqfdv virlnsapve gysehvgnkt tirmtypega
    121 plsdleyysn dlfvavlfks vdfnwlqamv kketlpfwvr lffwkqvaek iplqpkhfri
    181 lnpviiketa fdilqysepq srfwgrdknv ptigviavvl athlcdevsl agfgydlnqp
    241 rtplhyfdsq cmaamnfqtm hnvttetkfl lklvkegvvk dlsggidref
    Lactosylceramide alpha-2,3-sialyltransferase, isoform 4 NP_001341156.1,
    NP_001341158.1, NP_001341167.1 (SEQ ID NO: 389)
    1 mpseytyvkl rsdcsrpslq wytraqskmr rpslllkdil kctllvfgvw ilyilklnyt
    61 teecdmkkmh yvdpdhvkra qkyaqqvlqk ecrpkfakts mallfehrys vdllpfvqka
    121 pkdseaesky dppfgfrkfs skvqtllell pehdlpehlk aktcrrcvvi gsggilhgle
    181 lghtlnqfdv virinsapve gysehvgnkt tirmtypega plsdleyysn dlfvavlfks
    241 vdfnwlqamv kketlpfwvr lffwkqvaek iplqpkhfri lnpviiketa fdilqysepq
    301 srfwgrdknv ptigviavvl athlcdevsl agfgydlnqp rtplhyfdsq cmaamnfqtm
    361 hnvttetkfl lklvkegvvk dlsggidref
    Lactosylceramide alpha-2,3-sialyltransferase, isoform 5 NP_001341176.1 (SEQ
    ID NO: 390)
    1 mtypegapls dleyysndlf vavlfksvdf nwlqamvkke tlpfwvrlff wkqvaekipl
    61 qpkhfrilnp viiketafdi lqysepqsrf wgrdknvpti gviavvlath lcdevslagf
    121 gydlnqprtp lhyfdsqcma amnfqtmhnv ttetkfllkl vkegvvkdls ggidref
    Alpha-N-acetylneuraminide alpha-2,8-sialyltransferase, isoform 1 NP_003025.1
    (SEQ ID NO: 391)
    1 mspcgrarrq tsrgamavla wkfprtrlpm gasalcvvvl cwlyifpvyr lpnekeivqg
    61 vlqqgtawrr nqtaarafrk qmedccdpah lfamtkmnsp mgksmwydge flysftidns
    121 tyslfpqatp fqlplkkcav vgnggilkks gcgrqidean fvmrcnlppl sseytkdvgs
    181 ksqlvtanps iirqrfqnll wsrktfvdnm kiynhsyiym pafsmktgte pslrvyytls
    241 dvganqtvlf anpnflrsig kfwksrgiha krlstglflv saalglceev aiygfwpfsv
    301 nmheqpishh yydnvlpfsg fhampeeflq lwylhkigal rmqldpcedt slqpts
    Alpha-N-acetylneuraminide alpha-2,8-sialyltransferase, isoform 2
    NP_001291379.1 (SEQ ID NO: 392)
    1 mtgsfythsp ltiqltlssh rcnlpplsse ytkdvgsksq lvtanpsiir qrfqnllwsr
    61 ktfvdnmkiy nhsyiympaf smktgtepsl rvyytlsdvg anqtvlfanp nflrsigkfw
    121 ksrgihakrl stglflvsaa lglceevaiy gfwpfsvnmh eqpishhyyd nvlpfsgfha
    181 mpeeflqlwy lhkigalrmq ldpcedtslq pts
    Survivin, isoform 1 NP_001159.2 (SEQ ID NO: 393)
    1 mgaptlppaw qpflkdhris tfknwpfleg cactpermae agfihcpten epdlaqcffc
    61 fkelegwepd ddpieehkkh ssgcaflsvk kqfeeltlge flkldrerak nkiaketnnk
    121 kkefeetaek vrraieqlaa md
    Survivin, isoform 2 NP_001012270.1 (SEQ ID NO: 394)
    1 mgaptlppaw qpflkdhris tfknwpfleg cactpermae agfihcpten epdlaqcffc
    61 fkelegwepd ddpmqrkpti rrknlrklrr kcavpssswl pwieasgrsc lvpewlhhfq
    121 glfpgatslp vgplams
    Survivin, isoform 3 NP_001012271.1 (SEQ ID NO: 395)
    1 mgaptlppaw qpflkdhris tfknwpfleg cactpermae agfihcpten epdlaqcffc
    61 fkelegwepd ddpigpgtva yacntstlgg rggritreeh kkhssgcafl svkkqfeelt
    121 lgeflkldre raknkiaket nnkkkefeet aekvrraieq laamd
    T-box 4, isoform 1 NP_001308049.1 (SEQ ID NO: 396)
    1 mlqdkglses eeafrapgpa lgeasaanap epalaapgls gaalgsppgp gadvvaaaaa
    61 eqtienikvg lhekelwkkf heagtemiit kagrrmfpsy kvkvtgmnpk tkyillidiv
    121 paddhrykfc dnkwmvagka epampgrlyv hpdspatgah wmrqlvsfqk lkltnnhldp
    181 fghiilnsmh kyqprlhivk adennafgsk ntafcthvfp etsfisvtsy qnhkitqlki
    241 ennpfakgfr gsddsdlrva rlqskeypvi sksimrqrli spqlsatpdv gpllgthqal
    301 qhyqhengah sqlaepqdlp lstfptqrds slfyhclkrr adgtrhldlp ckrsyleaps
    361 svgedhyfrs pppydqqmls psycsevtpr eacmysgsgp eiagvsgvdd lpppplscnm
    421 wtsyspytsy svqtmetvpy qpfpthftat tmmprlptls aqssqppgna hfsvynqlsq
    481 sqvrergpsa sfprerglpq gcerkppsph lnaaneflys qtfslsress lqyhsgmgtv
    541 enwtdg
    T-box 4, isoform 2 NP_ 060958.2 (SEQ ID NO: 397)
    1 mlqdkglses eeafrapgpa lgeasaanap epalaapgls gaalgsppgp gadvvaaaaa
    61 eqtienikvg lhekelwkkf heagtemiit kagrrmfpsy kvkvtgmnpk tkyillidiv
    121 paddhrykfc dnkwmvagka epampgrlyv hpdspatgah wmrqlvsfqk lkltnnhldp
    181 fghiilnsmh kyqprlhivk adennafgsk ntafcthvfp etsfisvtsy qnhkitqlki
    241 ennpfakgfr gsddsdlrva rlqskeypvi sksimrqrli spqlsatpdv gpllgthqal
    301 qhyqhengah sqlaepqdlp lstfptqrds slfyhclkrr dgtrhldlpc krsyleapss
    361 vgedhyfrsp ppydqqmlsp sycsevtpre acmysgsgpe iagvsgvddl pppplscnmw
    421 tsyspytsys vqtmetvpyq pfpthftatt mmprlptlsa qssqppgnah fsvynqlsgs
    481 qvrergpsas fprerglpqg cerkppsphl naaneflysq tfslsressl qyhsgmgtve
    541 nwtdg
    Angiopoietin-1 receptor, isoform 1 NP_000450.2 (SEQ ID NO: 398)
    1 mdslaslvlc gvslllsgtv egamdlilin slplvsdaet sltciasgwr phepitigrd
    61 fealmnqhqd plevtqdvtr ewakkvvwkr ekaskingay fcegrvrgea irirtmkmrq
    121 qasflpatlt mtvdkgdnvn isfkkvlike edaviykngs fihsvprhev pdilevhlph
    181 aqpqdagvys aryiggnlft saftrlivrr ceaqkwgpec nhlctacmnn gvchedtgec
    241 icppgfmgrt cekacelhtf grtckercsg qegcksyvfc lpdpygcsca tgwkglqcne
    301 achpgfygpd cklrcscnng emcdrfqgcl cspgwqglqc eregiprmtp kivdlpdhie
    361 vnsgkfnpic kasgwplptn eemtivkpdg tvlhpkdfnh tdhfsvaift ihrilppdsg
    421 vwvcsvntva gmvekpfnis vkvlpkpina pnvidtghnf avinissepy fgdgpikskk
    481 llykpvnhye awqhiqvtne ivtlnylepr teyelcvqlv rrgeggeghp gpvrrfttas
    541 iglppprgln llpksqttln ltwqpifpss eddfyvever rsvqksdqqn ikvpgnitsv
    601 llnnlhpreq yvvrarvntk aqgewsedlt awtlsdilpp qpenikisni thssaviswt
    661 ildgysissi tirykvqgkn edqhvdvkik natitqyqlk glepetayqv difaennigs
    721 snpafshelv tlpesqapad lgggkmllia ilgsagmtcl tvllafliil qlkranvqrr
    781 maqafqnvre epavqfnsgt lalnrkvknn pdptiypvld wndikfqdvi gegnfgqvlk
    841 arikkdglrm daaikrmkey askddhrdfa gelevlcklg hhpniinllg acehrgylyl
    901 aieyaphgnl ldflrksrvl etdpafaian stastlssqq llhfaadvar gmdylsqkqf
    961 ihrdlaarni lvgenyvaki adfglsrgqe vyvkktmgrl pvrwmaiesl nysvyttnsd
    1021 vwsygvllwe ivslggtpyc gmtcaelyek lpqgyrlekp lncddevydl mrqcwrekpy
    1081 erpsfaqilv slnrmleerk tyvnttlyek ftyagidcsa eeaa
    Angiopoietin-1 receptor, isoform 2 NP_001277006.1 (SEQ ID NO: 399)
    1 mdslaslvlc gvslllsgtv egamdlilin slplvsdaet sltciasgwr phepitigrd
    61 fealmnqhqd plevtqdvtr ewakkvvwkr ekaskingay fcegrvrgea irirtmkmrq
    121 qasflpatlt mtvdkgdnvn isfkkvlike edaviykngs fihsvprhev pdilevhlph
    181 aqpqdagvys aryiggnlft saftrlivrr ceaqkwgpec nhlctacmnn gvchedtgec
    241 icppgfmgrt cekacelhtf grtckercsg qegcksyvfc lpdpygcsca tgwkglqcne
    301 giprmtpkiv dlpdhievns gkfnpickas gwplptneem tivkpdgtvl hpkdfnhtdh
    361 fsvaiftihr ilppdsgvwv csvntvagmv ekpfnisvkv lpkpinapnv idtghnfavi
    421 nissepyfgd gpikskklly kpvnhyeawq hiqvtneivt lnyleprtey elcvqlvrrg
    481 eggeghpgpv rrfttasigl ppprglnllp ksqttlnitw qpifpssedd fyveverrsv
    541 qksdqqnikv pgnltsvlln nlhpreqyvv rarvntkaqg ewsedltawt lsdilppqpe
    601 nikisniths saviswtild gysissitir ykvqgknedq hvdvkiknat itqyqlkgle
    661 petayqvdif aennigssnp afshelvtlp esqapadlgg gkmlliailg sagmtcltvl
    721 lafliilqlk ranvqrrmaq afqnvreepa vqfnsgtlal nrkvknnpdp tiypvldwnd
    781 ikfqdvigeg nfgqvlkari kkdglrmdaa ikrmkeyask ddhrdfagel evlcklghhp
    841 niinllgace hrgylylaie yaphgnlldf lrksrvletd pafaiansta stlssqqllh
    901 faadvargmd ylsqkqfihr dlaarnilvg enyvakiadf glsrgqevyv kktmgrlpvr
    961 wmaieslnys vyttnsdvws ygvllweivs lggtpycgmt caelyeklpq gyrlekpinc
    1021 ddevydlmrq cwrekpyerp sfaqilvsln rmleerktyv nttlyekfty agidcsaeea
    1081 a
    Angiopoietin-1 receptor, isoform 3 NP_001277007.1 (SEQ ID NO: 400)
    1 mdslaslvlc gvslllsasf lpatltmtvd kgdnvnisfk kvlikeedav iykngsfihs
    61 vprhevpdil evhlphaqpq dagvysaryi ggnlftsaft rlivrrceaq kwgpecnhlc
    121 tacmnngvch edtgecicpp gfmgrtceka celhtfgrtc kercsgqegc ksyvfclpdp
    181 ygcscatgwk glqcnegipr mtpkivdlpd hievnsgkfn pickasgwpl ptneemtivk
    241 pdgtvlhpkd fnhtdhfsva iftihrilpp dsgvwvcsvn tvagmvekpf nisvkvlpkp
    301 lnapnvidtg hnfaviniss epyfgdgpik skkllykpvn hyeawqhiqv tneivtlnyl
    361 eprteyelcv qlvrrgegge ghpgpvrrft tasiglpppr glnllpksqt tlnitwqpif
    421 psseddfyve verrsvqksd qqnikvpgnl tsvllnnlhp reqyvvrary ntkaqgewse
    481 dltawtlsdi lppqpeniki snithssavi swtildgysi ssitirykvq gknedqhvdv
    541 kiknatitqy qlkglepeta yqvdifaenn igssnpafsh elvtlpesqa padlgggkml
    601 liailgsagm tcltvllafl iilqlkranv qrrmaqafqn reepavqfns gtlalnrkvk
    661 nnpdptiypv ldwndikfqd vigegnfgqv lkarikkdgl rmdaaikrmk eyaskddhrd
    721 fagelevlck lghhpniinl lgacehrgyl ylaieyaphg nlldflrksr vletdpafai
    781 anstastlss qqllhfaadv argmdylsqk qfihrdlaar nilvgenyva kiadfglsrg
    841 qevyvkktmg rlpvrwmaie slnysvyttn sdvwsygvll weivslggtp ycgmtcaely
    901 eklpqgyrle kpincddevy dlmrqcwrek pyerpsfaqi lvslnrmlee rktyvnttly
    961 ekftyagidc saeeaa
    Telomerase reverse transcriptase, isoform 1 NP_937983.2 (SEQ ID NO: 401)
    1 mpraprcrav rsllrshyre vlplatfvrr lgpqgwrlvq rgdpaafral vaqclvcvpw
    61 darpppaaps frqvsclkel varvlqrlce rgaknvlafg falldgargg ppeafttsvr
    121 sylpntvtda lrgsgawgll lrrvgddvlv hllarcalfv lvapscayqv cgpplyqlga
    181 atqarpppha sgprrrlgce rawnhsvrea gvplglpapg arrrggsasr slplpkrprr
    241 gaapepertp vgqgswahpg rtrgpsdrgf cvvsparpae eatslegals gtrhshpsvg
    301 rqhhagppst srpprpwdtp cppvyaetkh flyssgdkeq lrpsfllssl rpsltgarrl
    361 vetiflgsrp wmpgtprrlp rlpqrywqmr plflellgnh aqcpygvllk thcplraavt
    421 paagvcarek pqgsvaapee edtdprrlvq llrghsspwq vygfvraclr rlvppglwgs
    481 rhnerrflrn tkkfislgkh aklslqeltw kmsvrdcawl rrspgvgcvp aaehrlreei
    541 lakflhwlms vyvvellrsf fyvtettfqk nrlffyrksv wsklqsigir qhlkrvqlre
    601 lseaevrqhr earpalltsr lrfipkpdgl rpivnmdyvv gartfrrekr aerltsrvka
    661 lfsvinyera rrpgllgasv lglddihraw rtfvlrvraq dpppelyfvk vdvtgaydti
    721 pqdrltevia siikpqntyc vrryavvqka ahghvrkafk shvstltdlq pymrqfvahl
    781 qetsplrdav vieqssslne assglfdvfl rfmchhavri rgksyvqcqg ipqgsilstl
    841 lcslcygdme nklfagirrd glllrlvddf llvtphltha ktflrtivrg vpeygcvvnl
    901 rktvvnfpve dealggtafv qmpahglfpw cgllldtrtl evqsdyssya rtsirasltf
    961 nrgfkagrnm rrklfgvlrl kchslfldlq vnslqtvctn iykilllqay rfhacvlqlp
    1021 fhqqvwknpt fflrvisdta slcysilkak nagmslgakg aagplpseav qwlchqafll
    1081 kltrhrvtyv pllgslrtaq tqlsrklpgt tltaleaaan palpsdfkti ld
    Telomerase reverse transcriptase, isoform 2 NP_001180305.1 (SEQ ID NO: 402)
    1 mpraprcrav rsllrshyre vlplatfvrr lgpqgwrlvq rgdpaafral vaqclvcvpw
    61 darpppaaps frqvsclkel varvlqrlce rgaknvlafg falldgargg ppeafttsvr
    121 sylpntvtda lrgsgawgll lrrvgddvlv hllarcalfv lvapscayqv cgpplyqlga
    181 atqarpppha sgprrrlgce rawnhsvrea gvplglpapg arrrggsasr slplpkrprr
    241 gaapepertp vgqgswahpg rtrgpsdrgf cvvsparpae eatslegals gtrhshpsvg
    301 rqhhagppst srpprpwdtp cppvyaetkh flyssgdkeq lrpsfllssl rpsltgarrl
    361 vetiflgsrp wmpgtprrlp rlpqrywqmr plflellgnh aqcpygvllk thcplraavt
    421 paagvcarek pqgsvaapee edtdprrlvq llrqhsspwq vygfvraclr rlvppglwgs
    481 rhnerrflrn tkkfislgkh aklslqeltw kmsvrdcawl rrspgvgcvp aaehrlreei
    541 lakflhwlms vyvvellrsf fyvtettfqk nrlffyrksv wsklqsigir qhlkrvqlre
    601 lseaevrqhr earpalltsr lrfipkpdgl rpivnmdyvv gartfrrekr aerltsrvka
    661 lfsvinyera rrpgllgasv lglddihraw rtfvlrvraq dpppelyfvk vdvtgaydti
    721 pqdrltevia siikpqntyc vrryavvqka ahghvrkafk shvstltdlq pymrqfvahl
    781 qetsplrdav vieqssslne assglfdvfl rfmchhavri rgksyvqcqg ipqgsilstl
    841 lcslcygdme nklfagirrd glllrlvddf llvtphltha ktflsyarts irasltfnrg
    901 fkagrnmrrk lfgvlrlkch slfldlqvns lqtvctniyk illlqayrfh acvlqlpfhq
    961 qvwknptffl rvisdtaslc ysilkaknag mslgakgaag plpseavqwl chqafllklt
    1021 rhrvtyvpll gslrtaqtql srklpgttlt aleaaanpal psdfktild
    Cellular tumor antigen p53, isoform a NP_000537.3, NP_00lll9584.1 (SEQ ID NO:
    403)
    1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp
    61 deaprmpeaa ppvapapaap tpaapapaps wplsssvpsq ktyqgsygfr lgflhsgtak
    121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt evvrrcphhe
    181 rcsdsdglap pqhlirvegn lrveylddrn tfrhsvvvpy eppevgsdct tihynymcns
    241 scmggmnrrp iltiitleds sgnllgrnsf evrvcacpgr drrteeenlr kkgephhelp
    301 pgstkralpn ntssspqpkk kpldgeyftl qirgrerfem frelnealel kdaqagkepg
    361 gsrahsshlk skkgqstsrh kklmfktegp dsd
    Cellular tumor antigen p53, isoform b NP_00lll9586.1 (SEQ ID NO: 404)
    1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp
    61 deaprmpeaa ppvapapaap tpaapapaps wplsssvpsq ktyqgsygfr lgflhsgtak
    121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt evvrrcphhe
    181 rcsdsdglap pqhlirvegn lrveylddrn tfrhsvvvpy eppevgsdct tihynymcns
    241 scmggmnrrp iltiitleds sgnllgrnsf evrvcacpgr drrteeenlr kkgephhelp
    301 pgstkralpn ntssspqpkk kpldgeyftl qdqtsfqken c
    Cellular tumor antigen p53, isoform c NP_00lll9585.1 (SEQ ID NO: 405)
    1 meepqsdpsv epplsqetfs dlwkllpenn vlsplpsqam ddlmlspddi eqwftedpgp
    61 deaprmpeaa ppvapapaap tpaapapaps wplsssvpsq ktyqgsygfr lgflhsgtak
    121 svtctyspal nkmfcqlakt cpvqlwvdst pppgtrvram aiykqsqhmt evvrrcphhe
    181 rcsdsdglap pqhlirvegn lrveylddrn tfrhsvvvpy eppevgsdct tihynymcns
    241 scmggmnrrp iltiitleds sgnllgrnsf evrvcacpgr drrteeenlr kkgephhelp
    301 pgstkralpn ntssspqpkk kpldgeyftl qmlldlrwcy flinss
    Cellular tumor antigen p53, isoform d NP_00lll9587.1 (SEQ ID NO: 406)
    1 mfcqlaktcp vqlwvdstpp pgtrvramai ykqsqhmtev vrrcphherc sdsdglappq
    61 hlirvegnlr veylddrntf rhsvvvpyep pevgsdctti hynymcnssc mggmnrrpil
    121 tiitledssg nllgrnsfev rvcacpgrdr rteeenlrkk gephhelppg stkralpnnt
    181 ssspqpkkkp ldgeyftlqi rgrerfemfr elnealelkd aqagkepggs rahsshlksk
    241 kgqstsrhkk lmfktegpds d
    Cellular tumor antigen p53, isoform e NP_00lll9588.1 (SEQ ID NO: 407)
    1 mfcqlaktcp vqlwvdstpp pgtrvramai ykqsqhmtev vrrcphherc sdsdglappq
    61 hlirvegnlr veylddrntf rhsvvvpyep pevgsdctti hynymcnssc mggmnrrpil
    121 tiitledssg nllgrnsfev rvcacpgrdr rteeenlrkk gephhelppg stkralpnnt
    181 ssspqpkkkp ldgeyftlqd qtsfqkenc
    Cellular tumor antigen p53, isoform f NP_00lll9589.1 (SEQ ID NO: 408)
    1 mfcqlaktcp vqlwvdstpp pgtrvramai ykqsqhmtev vrrcphherc sdsdglappq
    61 hlirvegnlr veylddrntf rhsvvvpyep pevgsdctti hynymcnssc mggmnrrpil
    121 tiitledssg nllgrnsfev rvcacpgrdr rteeenlrkk gephhelppg stkralpnnt
    181 ssspqpkkkp ldgeyftlqm lldlrwcyfl inss
    Cellular tumor antigen p53, isoform g NP_00lll9590.1, NP_001263689.1,
    NP_001263690.1 (SEQ ID NO: 409)
    1 mddlmlspdd ieqwftedpg pdeaprmpea appvapapaa ptpaapapap swplsssvps
    61 qktyqgsygf rlgflhsgta ksvtctyspa lnkmfcqlak tcpvqlwvds tpppgtrvra
    121 maiykqsqhm tevvrrcphh ercsdsdgla ppqhlirveg nlrveylddr ntfrhsvvvp
    181 yeppevgsdc ttihynymcn sscmggmnrr piltiitled ssgnllgrns fevrvcacpg
    241 rdrrteeenl rkkgephhel ppgstkralp nntssspqpk kkpldgeyft lqirgrerfe
    301 mfrelneale lkdaqagkep ggsrahsshl kskkgqstsr hkklmfkteg pdsd
    Cellular tumor antigen p53, isoform h NP_001263624.1 (SEQ ID NO: 410)
    1 mddlmlspdd ieqwftedpg pdeaprmpea appvapapaa ptpaapapap swplsssvps
    61 qktyqgsygf rlgflhsgta ksvtctyspa lnkmfcqlak tcpvqlwvds tpppgtrvra
    121 maiykqsqhm tevvrrcphh ercsdsdgla ppqhlirveg nlrveylddr ntfrhsvvvp
    181 yeppevgsdc ttihynymcn sscmggmnrr piltiitled ssgnllgrns fevrvcacpg
    241 rdrrteeenl rkkgephhel ppgstkralp nntssspqpk kkpldgeyft lqmlldlrwc
    301 yflinss
    Cellular tumor antigen p53, isoform i NP_001263625.1 (SEQ ID NO: 411)
    1 mddlmlspdd ieqwftedpg pdeaprmpea appvapapaa ptpaapapap swplsssvps
    61 qktyqgsygf rlgflhsgta ksvtctyspa lnkmfcqlak tcpvqlwvds tpppgtrvra
    121 maiykqsqhm tevvrrcphh ercsdsdgla ppqhlirveg nlrveylddr ntfrhsvvvp
    181 yeppevgsdc ttihynymcn sscmggmnrr piltiitled ssgnllgrns fevrvcacpg
    241 rdrrteeenl rkkgephhel ppgstkralp nntssspqpk kkpldgeyft lqdqtsfqke
    301 nc
    Cellular tumor antigen p53, isoform j NP_001263626.1 (SEQ ID NO: 412)
    1 maiykqsqhm tevvrrcphh ercsdsdgla ppqhlirveg nlrveylddr ntfrhsvvvp
    61 yeppevgsdc ttihynymcn sscmggmnrr piltiitled ssgnllgrns fevrvcacpg
    121 rdrrteeenl rkkgephhel ppgstkralp nntssspqpk kkpldgeyft lqirgrerfe
    181 mfrelneale lkdaqagkep ggsrahsshl kskkgqstsr hkklmfkteg pdsd
    Cellular tumor antigen p53, isoform k NP_001263627.1 (SEQ ID NO: 413)
    1 maiykqsqhm tevvrrcphh ercsdsdgla ppqhlirveg nlrveylddr ntfrhsvvvp
    61 yeppevgsdc ttihynymcn sscmggmnrr piltiitled ssgnllgrns fevrvcacpg
    121 rdrrteeenl rkkgephhel ppgstkralp nntssspqpk kkpldgeyft lqdqtsfqke
    181 nc
    Cellular tumor antigen p53, isoform 1 NP_001263628.1 (SEQ ID NO: 414)
    1 maiykqsqhm tevvrrcphh ercsdsdgla ppqhlirveg nlrveylddr ntfrhsvvvp
    61 yeppevgsdc ttihynymcn sscmggmnrr piltiitled ssgnllgrns fevrvcacpg
    121 rdrrteeenl rkkgephhel ppgstkralp nntssspqpk kkpldgeyft lqmlldlrwc
    181 yflinss
    Dopachrome tautomerase, isoform 1 NP_001913.2 (SEQ ID NO: 415)
    1 msplwwgfll sclgckilpg aqgqfprvcm tvdslvnkec cprlgaesan vcgsqqgrgq
    61 ctevradtrp wsgpyilrnq ddrelwprkf fhrtckctgn fagyncgdck fgwtgpncer
    121 kkppvirqni hslspqereq flgaldlakk rvhpdyvitt qhwlgllgpn gtqpqfancs
    181 vydffvwlhy ysvrdtllgp grpyraidfs hqgpafvtwh ryhllclerd lqrlignesf
    241 alpywnfatg rnecdvctdq lfgaarpddp tlisrnsrfs swetvcdsld dynhlvtlcn
    301 gtyegllrrn qmgrnsmklp tlkdirdcls lqkfdnppff qnstfsfrna legfdkadgt
    361 ldsqvmslhn lvhsflngtn alphsaandp ifvvlhsftd aifdewmkrf nppadawpqe
    421 lapighnrmy nmvpffppvt neelfltsdq lgysyaidlp vsveetpgwp ttllvvmgtl
    481 valvglfvll aflqyrrlrk gytplmethl sskryteea
    Dopachrome tautomerase, isoform 2 NP_001123361.1 (SEQ ID NO: 416)
    1 msplwwgfll sclgckilpg aqgqfprvcm tvdslvnkec cprlgaesan vcgsqqgrgq
    61 ctevradtrp wsgpyilrnq ddrelwprkf fhrtckctgn fagyncgdck fgwtgpncer
    121 kkppvirgni hslspqereq flgaldlakk rvhpdyvitt qhwlgllgpn gtqpqfancs
    181 vydffvwlhy ysvrdtllgp grpyraidfs hqgpafvtwh ryhllclerd lqrlignesf
    241 alpywnfatg rnecdvctdq lfgaarpddp tlisrnsrfs swetvcdsld dynhlvtlcn
    301 gtyegllrrn qmgrnsmklp tlkdirdcls lqkfdnppff qnstfsfrna legfdkadgt
    361 ldsqvmslhn lvhsflngtn alphsaandp ifvvisnrll ynattnileh vrkekatkel
    421 pslhvlvlhs ftdaifdewm krfnppadaw pqelapighn rmynmvpffp pvtneelflt
    481 sdqlgysyai dlpvsveetp gwpttllvvm gtlvalvglf vllaflqyrr lrkgytplme
    541 thlsskryte ea
    Dopachrome tautomerase, isoform 3 NP_001309lll.1, NP_001309112.1,
    NP_001309113.1, NP_ 001309114.1 (SEQ ID NO: 417)
    1 mgrnsmklpt lkdirdclsl qkfdnppffq nstfsfrnal egfdkadgtl dsqvmslhnl
    61 vhsflngtna lphsaandpi fvvlhsftda ifdewmkrfn ppadawpqel apighnrmyn
    121 mvpffppvtn eelfltsdql gysyaidlpv sveetpgwpt tllvvmgtiv alvglfvlla
    181 flqyrrlrkg ytplmethls skryteea
    Dopachrome tautomerase, isoform 4, NP_001309115.1 (SEQ ID NO: 418)
    1 mllgiqrqmk crlrsdvtkr leedehvnth spmrrgnfag yncgdckfgw tgpncerkkp
    61 pvirqnihsl spqereqflg aldlakkrvh pdyvittqhw lgllgpngtq pqfancsvyd
    121 ffvwlhyysv rdtllgpgrp yraidfshqg pafvtwhryh llclerdlqr lignesfalp
    181 ywnfatgrne cdvctdqlfg aarpddptli srnsrfsswe tvcdslddyn hlvtlcngty
    241 egllrrnqmg rnsmklptlk dirdclslqk fdnppffqns tfsfrnaleg fdkadgtlds
    301 qvmslhnlvh sflngtnalp hsaandpifv vlhsftdaif dewmkrfnpp adawpgelap
    361 ighnrmynmv pffppvtnee lfltsdqlgy syaidlpvsv eetpgwpttl lvvmgtlval
    421 vglfvllafl qyrrlrkgyt plmethlssk ryteea
    Transformation/transcription domain associated protein, isoform 1
    NP_001231509.1 (SEQ ID NO: 419)
    1 mafvatqgat vvdqttlmkk ylqfvaaltd vntpdetklk mmqevsenfe nvtsspqyst
    61 flehiiprfl tflqdgevqf lqekpaqqlr klvleiihri ptnehlrpht knvlsvmfrf
    121 leteneenvl iclriiielh kqfrppitqe ihhfldfvkq iykelpkvvn ryfenpqvip
    181 entvpppemv gmittiavkv nperedsetr thsiiprgsl slkvlaelpi ivvlmyglyk
    241 lnihnvvaef vplimntiai qvsaqarghk lynkelyadf iaaqiktlsf layiiriyqe
    301 lvtkysqqmv kgmlqllsnc paetahlrke lliaakhilt telrnqfipc mdklfdesil
    361 igsgytaret lrplaystla dlvhhvrqhl plsdlslavq lfakniddes lpssiqtmsc
    421 klllnlvdci rskseqesgn grdvlmrmle vfvlkfhtia ryqlsaifkk ckpqselgav
    481 eaalpgvpta paapgpapsp apvpappppp pppppatpvt papvppfekq gekdkedkqt
    541 fqvtdcrslv ktivcgvkti twgitsckap geaqfipnkq lqpketqiyi klvkyamqal
    601 diyqvqiagn gqtyirvanc qtvrmkeeke vlehfagvft mmnpltfkei fqttvpymve
    661 risknyalqi vansflanpt tsalfatilv eylldrlpem gsnvelsnly lklfklvfgs
    721 vslfaaeneq mlkphlhkiv nssmelaqta kepynyflll ralfrsiggg shdllyqefl
    781 pllpnllqgl nmlqsglhkq hmkdlfvelc ltvpvrlssl lpylpmlmdp lvsalngsqt
    841 lvsqglrtle lcvdnlqpdf lydhiqpvra elmqalwrtl rnpadsishv ayrvlgkfgg
    901 snrkmlkesq klhyvvtevq gpsitvefsd ckaslqlpme kaietaldcl ksantepyyr
    961 rqawevikcf lvammsledn khalyqllah pnftektipn viishrykaq dtparktfeq
    1021 altgafmsav ikdlrpsalp fvaslirhyt mvavaqqcgp fllpcyqvgs qpstamfhse
    1081 engskgmdpl vlidaiaicm ayeekelcki gevalavifd vasiilgske racqlplfsy
    1141 iverlcaccy eqawyaklgg vvsikflmer lpltwvlqnq qtflkallfv mmdltgevsn
    1201 gavamakttl eqllmrcatp lkdeeraeei vaaqeksfhh vthdlvrevt spnstvrkqa
    1261 mhslqvlaqv tgksvtvime phkevlqdmv ppkkhllrhq panaqiglme gntfcttlqp
    1321 rlftmdlnvv ehkvfytell nlceaedsal tklpcykslp slvplriaal nalaacnylp
    1381 qsrekiiaal fkalnstnse lqeageacmr kflegatiev dqihthmrpl lmmlgdyrsl
    1441 tlnvvnrlts vtrlfpnsfn dkfcdqmmqh lrkwmevvvi thkggqrsdg nesisecgrc
    1501 plspfcqfee mkicsaiinl fhlipaapqt lvkpllevvm kteramliea gspfreplik
    1561 fltrhpsqtv elfmmeatln dpqwsrmfms flkhkdarpl rdvlaanpnr fitlllpgga
    1621 qtavrpgsps tstmrldlqf qaikiisiiv knddswlasq hslvsqlrrv wvsenfqerh
    1681 rkenmaatnw kepkllaycl lnyckrnygd iellfqllra ftgrflcnmt flkeymeeei
    1741 pknysiaqkr alffrfvdfn dpnfgdelka kvlqhilnpa flysfekgeg eqllgppnpe
    1801 gdnpesitsv fitkvldpek qadmldslri yllqyatllv ehaphhihdn nknrnsklrr
    1861 lmtfawpcll skacvdpack ysghlllahi iakfaihkki vlqvfhsllk ahamearaiv
    1921 rqamailtpa vparmedghq mlthwtrkii veeghtvpql vhilhlivqh fkvyypvrhh
    1981 lvqhmvsamq rlgftpsvti eqrrlavdls evvikwelqr ikdqqpdsdm dpnssgegvn
    2041 sysssikrgl svdsaqevkr frtatgaisa vfgrsqslpg adsllakpid kqhtdtvvnf
    2101 lirvacqvnd ntntagspge vlsrrcvnll ktalrpdmwp kselklqwfd kllmtveqpn
    2161 qvnygnictg levlsflltv lqspailssf kplqrgiaac mtcgntkvlr avhsllsrlm
    2221 sifptepsts svaskyeele clyaavgkvi yegltnyeka tnanpsqlfg tlmilksacs
    2281 nnpsyidrli svfmrslqkm vrehlnpqaa sgsteatsgt selvmlslel vktrlavmsm
    2341 emrknfiqai ltsliekspd akilravvki veewvknnsp maanqtptlr eksillvkmm
    2401 tyiekrfped lelnaqfldl vnyvyrdetl sgseltakle paflsglrca qplirakffe
    2461 vfdnsmkrrv yerllyvtcs qnweamgnhf wikqcielll avcekstpig tscqgamlps
    2521 itnvinlads hdraafamvt hvkqeprere nseskeedve idielapgdq tstpktkels
    2581 ekdignqlhm ltnrhdkfld tlrevktgal lsafvqlchi sttlaektwv qlfprlwkil
    2641 sdrqqhalag eispflcsgs hqvgrdcqps alncfveams qcvppipirp cvlkylgkth
    2701 nlwfrstlml ehqafekgls lqikpkqtte fyeqesitpp qqeildslae lysllqeedm
    2761 waglwqkrck ysetataiay eqhgffeqaq esyekamdka kkehersnas paifpeyqlw
    2821 edhwircske lnqwealtey gqskghinpy lvlecawrvs nwtamkealv qvevscpkem
    2881 awkvnmyrgy laichpeeqq lsfierlvem asslairewr rlphvvshvh tpllqaaqqi
    2941 ielqeaaqin aglqptnlgr nnslhdmktv vktwrnrlpi vsddlshwss ifmwrqhhyq
    3001 gkptwsgmhs ssivtayens sqhdpssnna mlgvhasasa iiqygkiark qglvnvaldi
    3061 lsrihtiptv pivdcfqkir qqvkcylqla gvmgknecmq gleviestnl kyftkemtae
    3121 fyalkgmfla qinkseeank afsaavqmhd vlvkawamwg dylenifvke rqlhlgvsai
    3181 tcylhacrhq nesksrkyla kvlwllsfdd dkntladavd kycigvppiq wlawipqllt
    3241 clvgsegkll lnlisqvgrv ypqavyfpir tlyltlkieq reryksdpgp iratapmwrc
    3301 srimhmqrel hptllssleg ivdqmvwfre nwheevlrql qqglakcysv afeksgavsd
    3361 akitphtlnf vkklvstfgv glenvsnvst mfssaasesl arraqataqd pvfqklkgqf
    3421 ttdfdfsvpg smklhnlisk lkkwikilea ktkqlpkffl ieekcrflsn fsaqtaevei
    3481 pgeflmpkpt hyyikiarfm prveivqkhn taarrlyirg hngkiypylv mndacltesr
    3541 reervlqllr llnpclekrk ettkrhlfft vprvvayspq mrlvednpss lslveiykqr
    3601 cakkgiehdn pisryydrla tvqargtqas hqvlrdilke vqsnmvprsm lkewalhtfp
    3661 natdywtfrk mftiqlalig faefvlhlnr lnpemlqiaq dtgklnvayf rfdindatgd
    3721 ldanrpvpfr ltpniseflt tigvsgplta smiavarcfa qpnfkvdgil ktvlrdeiia
    3781 whkktqedts splsaagqpe nmdsqqlvsl vqkavtaimt rlhnlaqfeg geskvntlva
    3841 aansldnlcr mdpawhpwl
    Transformation/transcription domain associated protein, isoform 2 NP_003487.1
    (SEQ ID NO: 420)
    1 mafvatqgat vvdqttlmkk ylqfvaaltd vntpdetklk mmqevsenfe nvtsspqyst
    61 flehiiprfl tflqdgevqf lqekpaqqlr klvleiihri ptnehlrpht knvlsvmfrf
    121 leteneenvl iclriiielh kqfrppitqe ihhfldfvkq iykelpkvvn ryfenpqvip
    181 entvpppemv gmittiavkv nperedsetr thsiiprgsl slkvlaelpi ivvlmyqlyk
    241 lnihnvvaef vplimntiai qvsaqarqhk lynkelyadf iaaqiktlsf layiiriyqe
    301 lvtkysqqmv kgmlqllsnc paetahlrke lliaakhilt telrnqfipc mdklfdesil
    361 igsgytaret lrplaystla dlvhhvrqhl plsdlslavq lfakniddes lpssiqtmsc
    421 klllnlvdci rskseqesgn grdvlmrmle vfvlkfhtia ryqlsaifkk ckpqselgav
    481 eaalpgvpta paapgpapsp apvpappppp pppppatpvt papvppfekq gekdkedkqt
    541 fqvtdcrslv ktivcgvkti twgitsckap geaqfipnkq lqpketqiyi klvkyamqal
    601 diyqvqiagn gqtyirvanc qtvrmkeeke vlehfagvft mmnpltfkei fqttvpymve
    661 risknyalqi vansflanpt tsalfatilv eylldrlpem gsnvelsnly lklfklvfgs
    721 vslfaaeneq mlkphlhkiv nssmelaqta kepynyflll ralfrsiggg shdllyqefl
    781 pllpnllqgl nmlqsglhkq hmkdlfvelc ltvpvrlssl lpylpmlmdp lvsalngsqt
    841 lvsqglrtle lcvdnlqpdf lydhiqpvra elmgalwrtl rnpadsishv ayrvlgkfgg
    901 snrkmlkesq klhyvvtevq gpsitvefsd ckaslqlpme kaietaldcl ksantepyyr
    961 rqawevikcf lvammsledn khalyqllah pnftektipn viishrykaq dtparktfeq
    1021 altgafmsav ikdlrpsalp fvaslirhyt mvavaqqcgp fllpcyqvgs qpstamfhse
    1081 engskgmdpl vlidaiaicm ayeekelcki gevalavifd vasiilgske racqlplfsy
    1141 iverlcaccy eqawyaklgg vvsikflmer lpltwvlqnq qtflkallfv mmdltgevsn
    1201 gavamakttl eqllmrcatp lkdeeraeei vaaqeksfhh vthdlvrevt spnstvrkqa
    1261 mhslqvlaqv tgksvtvime phkevlqdmv ppkkhllrhq panaqiglme gntfcttlqp
    1321 rlftmdlnvv ehkvfytell nlceaedsal tklpcykslp slvplriaal nalaacnylp
    1381 qsrekiiaal fkalnstnse lqeageacmr kflegatiev dqihthmrpl lmmlgdyrsl
    1441 tlnvvnrlts vtrlfpnsfn dkfcdqmmqh lrkwmevvvi thkggqrsdg nemkicsaii
    1501 nlfhlipaap qtlvkpllev vmkteramli eagspfrepl ikfltrhpsq tvelfmmeat
    1561 lndpqwsrmf msflkhkdar plrdvlaanp nrfitlllpg gaqtavrpgs pststmrldl
    1621 qfqaikiisi ivknddswla sqhslvsqlr rvwvsenfqe rhrkenmaat nwkepkllay
    1681 cllnyckrny gdiellfqll raftgrflcn mtflkeymee eipknysiaq kralffrfvd
    1741 fndpnfgdel kakvlqhiln paflysfekg egeqllgppn pegdnpesit svfitkvldp
    1801 ekqadmldsl riyllqyatl lvehaphhih dnnknrnskl rrlmtfawpc llskacvdpa
    1861 ckysghllla hiiakfaihk kivlqvfhsl lkahameara ivrqamailt pavparmedg
    1921 hqmlthwtrk iiveeghtvp qlvhilhliv qhfkvyypvr hhlvqhmvsa mqrlgftpsv
    1981 tieqrrlavd lsevvikwel qrikdqqpds dmdpnssgeg vnsysssikr glsvdsaqev
    2041 krfrtatgai savfgrsqsl pgadsllakp idkqhtdtvv nflirvacqv ndntntagsp
    2101 gevlsrrcvn llktalrpdm wpkselklqw fdkllmtveq pnqvnygnic tglevlsfll
    2161 tvlqspails sfkplqrgia acmtcgntkv lravhsllsr lmsifpteps tssvaskyee
    2221 leclyaavgk viyegltnye katnanpsql fgtlmilksa csnnpsyidr lisvfmrslq
    2281 kmvrehlnpq aasgsteats gtselvmlsl elvktrlavm smemrknfiq ailtslieks
    2341 pdakilravv kiveewvknn spmaanqtpt lreksillvk mmtyiekrfp edlelnaqfl
    2401 dlvnyvyrde tlsgseltak lepaflsglr caqplirakf fevfdnsmkr rvyerllyvt
    2461 csqnweamgn hfwikqciel llavcekstp igtscqgaml psitnvinla dshdraafam
    2521 vthvkqepre renseskeed veidielapg dqtstpktke lsekdignql hmltnrhdkf
    2581 ldtlrevktg allsafvqlc histtlaekt wvqlfprlwk ilsdrqqhal ageispflcs
    2641 gshqvqrdcq psalncfvea msqcvppipi rpcvlkylgk thnlwfrstl mlehqafekg
    2701 lslqikpkqt tefyeqesit ppqqeildsl aelysllqee dmwaglwqkr ckysetatai
    2761 ayeqhgffeq aqesyekamd kakkehersn aspaifpeyq lwedhwircs kelnqwealt
    2821 eygqskghin pylvlecawr vsnwtamkea lvqvevscpk emawkvnmyr gylaichpee
    2881 qqlsfierlv emasslaire wrrlphvvsh vhtpllqaaq qiielqeaaq inaglqptnl
    2941 grnnslhdmk tvvktwrnrl pivsddlshw ssifmwrqhh yqaivtayen ssqhdpssnn
    3001 amlgvhasas aiiqygkiar kqglvnvald ilsrihtipt vpivdcfqki rqqvkcylql
    3061 agvmgknecm qgleviestn lkyftkemta efyalkgmfl aqinkseean kafsaavqmh
    3121 dvlvkawamw gdylenifvk erqlhlgvsa itcylhacrh qnesksrkyl akvlwllsfd
    3181 ddkntladav dkycigvppi qwlawipqll tclvgsegkl llnlisqvgr vypqavyfpi
    3241 rtlyltlkie qreryksdpg piratapmwr csrimhmqre lhptllssle givdqmvwfr
    3301 enwheevlrq lqgglakcys vafeksgavs dakitphtln fvkklvstfg vglenvsnvs
    3361 tmfssaases larraqataq dpvfqklkgq fttdfdfsvp gsmklhnlis klkkwikile
    3421 aktkqlpkff lieekcrfls nfsaqtaeve ipgeflmpkp thyyikiarf mprveivqkh
    3481 ntaarrlyir ghngkiypyl vmndacltes rreervlqll rllnpclekr kettkrhlff
    3541 tvprvvaysp qmrlvednps slslveiykg rcakkgiehd npisryydrl atvqargtqa
    3601 shgvlrdilk evqsnmvprs mlkewalhtf pnatdywtfr kmftiqlali gfaefvlhln
    3661 rlnpemlqia qdtgklnvay frfdindatg dldanrpvpf rltpnisefl ttigvsgplt
    3721 asmiavarcf aqpnfkvdgi lktvlrdeii awhkktqedt ssplsaagqp enmdsqqlvs
    3781 lvqkavtaim trlhnlaqfe ggeskvntiv aaansldnlc rmdpawhpwl
    Tyrosinase precursor NP_000363.1 (SEQ ID NO: 421)
    1 mllavlycll wsfqtsaghf pracvssknl mekeccppws gdrspcgqls grgscqnill
    61 snaplgpqfp ftgvddresw psvfynrtcq csgnfmgfnc gnckfgfwgp ncterrllvr
    121 rnifdlsape kdkffayltl akhtissdyv ipigtygqmk ngstpmfndi niydlfvwmh
    181 yyvsmdallg gseiwrdidf aheapaflpw hrlfllrweq eiqkltgden ftipywdwrd
    241 aekcdictde ymggqhptnp nllspasffs swqivcsrle eynshqslcn gtpegplrrn
    301 pgnhdksrtp rlpssadvef clsltqyesg smdkaanfsf rntlegfasp ltgiadasqs
    361 smhnalhiym ngtmsqvqgs andpifllhh afvdsifeqw lrrhrplqev ypeanapigh
    421 nresymvpfi plyrngdffi sskdlgydys ylqdsdpdsf qdyiksyleq asriwswllg
    481 aamvgavlta llaglvsllc rhkrkqlpee kqpllmeked yhslyqshl
    Vascular endothelial growth factor A, isoform a NP_001020537.2 (SEQ ID NO:
    422)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep gpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvyvgar cclmpwslpg
    361 phpcgpcser rkhlfvqdpq tckcsckntd srckarqlel nertcrcdkp rr
    Vascular endothelial growth factor A, isoform b NP_003367.4 (SEQ ID NO: 423)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvpcgpc serrkhlfvq
    361 dpqtckcsck ntdsrckarq lelnertcrc dkprr
    Vascular endothelial growth factor A, isoform c NP_001020538.2 (SEQ ID NO:
    424)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksrp cgpcserrkh lfvqdpqtck
    361 csckntdsrc karqlelner tcrcdkprr
    Vascular endothelial growth factor A, isoform d NP_001020539.2 (SEQ ID NO:
    425)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckarqleln
    361 ertcrcdkpr r
    Vascular endothelial growth factor A, isoform e NP_001020540.2 (SEQ ID NO:
    426)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckm
    Vascular endothelial growth factor A, isoform f NP_001020541.2 (SEQ ID NO:
    427)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe kcdkprr
    Vascular endothelial growth factor A, isoform g NP_001028928.1 (SEQ ID NO:
    428)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckarqleln
    361 ertcrsltrk d
    Vascular endothelial growth factor A, isoform h NP_001165093.1 (SEQ ID NO:
    429)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rcdkprr
    Vascular endothelial growth factor A, isoform i NP_001165094.1 (SEQ ID NO:
    430)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvyvgar cclmpwslpg
    181 phpcgpcser rkhlfvqdpq tckcsckntd srckarqlel nertcrcdkp rr
    Vascular endothelial growth factor A, isoform j NP_001165095.1 (SEQ ID NO:
    431)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietlvd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvpcgpc serrkhlfvq
    181 dpqtckcsck ntdsrckarq lelnertcrc dkprr
    Vascular endothelial growth factor A, isoform k NP_001165096.1 (SEQ ID NO:
    432)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksrp cgpcserrkh lfvqdpqtck
    181 csckntdsrc karqlelner tcrcdkprr
    Vascular endothelial growth factor A, isoform 1 NP_001165097.1 (SEQ ID NO:
    433)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe npcgpcserr khlfvgdpqt ckcsckntds rckarqleln
    181 ertcrcdkpr r
    Vascular endothelial growth factor A, isoform m NP_001165098.1 (SEQ ID NO:
    434)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckm
    Vascular endothelial growth factor A, isoform n NP_001165099.1 (SEQ ID NO:
    435)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe kcdkprr
    Vascular endothelial growth factor A, isoform o NP_001165100.1 (SEQ ID NO:
    436)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckarqleln
    181 ertcrsltrk d
    Vascular endothelial growth factor A, isoform p NP_001165101.1 (SEQ ID NO:
    437)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rcdkprr
    Vascular endothelial growth factor A, isoform q NP_001191313.1 (SEQ ID NO:
    438)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvcdkpr r
    Vascular endothelial growth factor A, isoform r NP_001191314.1 (SEQ ID NO:
    439)
    1 mtdrqtdtap spsyhllpgr rrtvdaaasr gqgpepapgg gvegvgargv alklfvqllg
    61 csrfggavvr ageaepsgaa rsassgreep qpeegeeeee keeergpqwr lgarkpgswt
    121 geaavcadsa paarapqala rasgrggrva rrgaeesgpp hspsrrgsas ragpgraset
    181 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    241 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    301 sflqhnkcec rpkkdrarqe kksvrgkgkg qkrkrkksry kswsvcdkpr r
    Vascular endothelial growth factor A, isoform s NP_001273973.1 (SEQ ID NO:
    440)
    1 maegggqnhh evvkfmdvyq rsychpietl vdifqeypde ieyifkpscv plmrcggccn
    61 deglecvpte esnitmqimr ikphqgqhig emsflqhnkc ecrpkkdrar genpcgpcse
    121 rrkhlfvqdp qtckcscknt dsrckarqle lnertcrcdk prr
    Vascular endothelial growth factor A, isoform VEGF-Ax precursor
    NP_001303939.1 (SEQ ID NO: 441)
    1 mnfllswvhw slalllylhh akwsqaapma egggqnhhev vkfmdvyqrs ychpietivd
    61 ifqeypdeie yifkpscvpl mrcggccnde glecvptees nitmqimrik phqgqhigem
    121 sflqhnkcec rpkkdrarqe npcgpcserr khlfvqdpqt ckcsckntds rckarqleln
    181 ertcrcdkpr rsagqeegas lrvsgtrslt rkd
    WD repeat-containing protein 46, isoform 1 NP_005443.3 (SEQ ID NO: 442)
    1 metapkpgkd vppkkdklqt krkkprrywe eetvpttaga spgpprnkkn relrpqrpkn
    61 ayilkksris kkpqvpkkpr ewknpesqrg lsgtqdpfpg papvpvevvq kfcridksrk
    121 lphskaktrs rlevaeaeee etsikaarse lllaeepgfl egedgedtak icqadiveav
    181 diasaakhfd lnlrqfgpyr lnysrtgrhl afggrrghva aldwvtkklm ceinvmeavr
    241 dirflhseal lavaqnrwlh iydnqgielh cirrcdrvtr leflpfhfll atasetgflt
    301 yldvsvgkiv aalnaragrl dvmsqnpyna vihlghsngt vslwspamke plakilchrg
    361 gvravavdst gtymatsgld hqlkifdlrg tyqplstrtl phgaghlafs qrgllvagmg
    421 dvvniwagqg kasppsleqp ylthrlsgpv hglqfcpfed vlgvghtggi tsmlvpgage
    481 pnfdglesnp yrsrkgrqew evkallekvp aelicldpra laevdvisle qgkkeqierl
    541 gydpqakapf qpkpkqkgrs staslvkrkr kvmdeehrdk vrqslqqqhh keakakptga
    601 rpsaldrfvr
    WD repeat-containing protein 46, isoform 2 NP_001157739.1 (SEQ ID NO: 443)
    1 metapkpgkd vppkkdklqt krkkprewkn pesqrglsgt qdpfpgpapv pvevvqkfcr
    61 idksrklphs kaktrsrlev aeaeeeetsi kaarsellla eepgfleged gedtakicqa
    121 diveavdias aakhfdlnlr qfgpyrlnys rtgrhlafgg rrghvaaldw vtkklmcein
    181 vmeavrdirf lhseallava qnrwlhiydn qgielhcirr cdrvtrlefl pfhfllatas
    241 etgfltyldv svgkivaaln aragrldvms qnpynavihl ghsngtvslw spamkeplak
    301 ilchrggvra vavdstgtym atsgldhqlk ifdlrgtyqp lstrtlphga ghlafsqrgl
    361 lvagmgdvvn iwagqgkasp psleqpylth rlsgpvhglq fcpfedvlgv ghtggitsml
    421 vpgagepnfd glesnpyrsr kqrqewevka llekvpaeli cldpralaev dvisleqgkk
    481 eqierlgydp qakapfqpkp kqkgrsstas lvkrkrkvmd eehrdkvrqs lqqqhhkeak
    541 akptgarpsa ldrfvr
    Wilms tumor protein, isoform A NP_000369.4 (SEQ ID NO: 444)
    1 mdflllqdpa stcvpepasq htlrsgpgcl qqpeqqgvrd pggiwaklga aeasaerlqg
    61 rrsrgasgse pqqmgsdvrd lnallpavps lgggggcalp vsgaaqwapv ldfappgasa
    121 ygslggpapp papppppppp phsfikqeps wggaepheeq clsaftvhfs gqftgtagac
    181 rygpfgpppp sqassgqarm fpnapylpsc lesqpairnq gystvtfdgt psyghtpshh
    241 aaqfpnhsfk hedpmgqqgs lgeqqysvpp pvygchtptd sctgsqalll rtpyssdnly
    301 qmtsqlecmt wnqmnlgatl kghstgyesd nhttpilcga qyrihthgvf rgiqdvrrvp
    361 gvaptlvrsa setsekrpfm caypgcnkry fklshlqmhs rkhtgekpyq cdfkdcerrf
    421 srsdqlkrhq rrhtgvkpfq cktcqrkfsr sdhlkthtrt htgekpfscr wpscqkkfar
    481 sdelvrhhnm hqrnmtklql al
    Wilms tumor protein, isoform B NP_077742.3 (SEQ ID NO: 445)
    1 mdflllqdpa stcvpepasq htlrsgpgcl qqpeqqgvrd pggiwaklga aeasaerlqg
    61 rrsrgasgse pqqmgsdvrd lnallpavps lgggggcalp vsgaaqwapv ldfappgasa
    121 ygslggpapp papppppppp phsfikqeps wggaepheeq clsaftvhfs gqftgtagac
    181 rygpfgpppp sqassgqarm fpnapylpsc lesqpairnq gystvtfdgt psyghtpshh
    241 aaqfpnhsfk hedpmgqqgs lgeqqysvpp pvygchtptd sctgsgalll rtpyssdnly
    301 qmtsqlecmt wnqmnlgatl kgvaagssss vkwtegqsnh stgyesdnht tpilcgaqyr
    361 ihthgvfrgi qdvrrvpgva ptivrsaset sekrpfmcay pgcnkryfkl shlqmhsrkh
    421 tgekpyqcdf kdcerrfsrs dqlkrhgrrh tgvkpfqckt cqrkfsrsdh lkthtrthtg
    481 ekpfscrwps cqkkfarsde lvrhhnmhqr nmtklqlal
    Wilms tumor protein, isoform D NP_077744.4 (SEQ ID NO: 446)
    1 mdflllqdpa stcvpepasq htlrsgpgcl qqpeqqgvrd pggiwaklga aeasaerlqg
    61 rrsrgasgse pqqmgsdvrd lnallpavps lgggggcalp vsgaaqwapv ldfappgasa
    121 ygslggpapp papppppppp phsfikqeps wggaepheeq clsaftvhfs gqftgtagac
    181 rygpfgpppp sqassgqarm fpnapylpsc lesqpairnq gystvtfdgt psyghtpshh
    241 aaqfpnhsfk hedpmgqqgs lgeqqysvpp pvygchtptd sctgsqalll rtpyssdnly
    301 qmtsqlecmt wnqmnlgatl kgvaagssss vkwtegqsnh stgyesdnht tpilcgaqyr
    361 ihthgvfrgi qdvrrvpgva ptivrsaset sekrpfmcay pgcnkryfkl shlqmhsrkh
    421 tgekpyqcdf kdcerrfsrs dqlkrhqrrh tgvkpfqckt cqrkfsrsdh lkthtrthtg
    481 ktsekpfscr wpscqkkfar sdelvrhhnm hqrnmtklql al
    Wilms tumor protein, isoform E NP_001185480.1 (SEQ ID NO: 447)
    1 mekgystvtf dgtpsyghtp shhaaqfpnh sfkhedpmgq qgslgeqqys vpppvygcht
    61 ptdsctgsqa lllrtpyssd nlyqmtsqle cmtwnqmnlg atlkgvaags sssvkwtegq
    121 snhstgyesd nhttpilcga qyrihthgvf rgiqdvrrvp gvaptlvrsa setsekrpfm
    181 caypgcnkry fklshlqmhs rkhtgekpyq cdfkdcerrf srsdqlkrhq rrhtgvkpfq
    241 cktcqrkfsr sdhlkthtrt htgekpfscr wpscqkkfar sdelvrhhnm hqrnmtklql
    301 al
    Wilms tumor protein, isoform F NP_001185481.1 (SEQ ID NO: 448)
    1 mekgystvtf dgtpsyghtp shhaaqfpnh sfkhedpmgq qgslgeqqys vpppvygcht
    61 ptdsctgsqa lllrtpyssd nlyqmtsqle cmtwnqmnlg atlkghstgy esdnhttpil
    121 cgaqyrihth gvfrgiqdvr rvpgvaptiv rsasetsekr pfmcaypgcn kryfklshlq
    181 mhsrkhtgek pyqcdfkdce rrfsrsdqlk rhqrrhtgvk pfqcktcqrk fsrsdhlkth
    241 trthtgktse kpfscrwpsc qkkfarsdel vrhhnmhqrn mtklqlal
    X antigen family member 1, isoform a NP_001091063.2 (SEQ ID NO: 449)
    1 mespkkknqq lkvgilhlgs rqkkiriqlr sqcatwkvic kscisqtpgi nldlgsgvkv
    61 kiipkeehck mpeageeqpq v
    X antigen family member 1, isoform d NP_001091065.1 (SEQ ID NO: 450)
    1 mespkkknqq lkvgilhlgs rqkkiriqlr sqvlgremrd megdlqelhq sntgdksgfg
    61 frrqgednt
    X-linked inhibitor of apoptosis NP_001158.2, NP_001191330.1 (SEQ ID NO: 451)
    1 mtfnsfegsk tcvpadinke eefveefnrl ktfanfpsgs pvsastlara gflytgegdt
    61 vrcfschaav drwqygdsav grhrkvspnc rfingfylen satqstnsgi qngqykveny
    121 lgsrdhfald rpsethadyl lrtgqvvdis dtiyprnpam yseearlksf qnwpdyahlt
    181 prelasagly ytgigdqvqc fccggklknw epcdrawseh rrhfpncffv lgrnlnirse
    241 sdavssdrnf pnstnlprnp smadyearif tfgtwiysvn keqlaragfy algegdkvkc
    301 fhcgggltdw kpsedpweqh akwypgckyl leqkgqeyin nihlthslee clvrttektp
    361 sltrriddti fqnpmvqeai rmgfsfkdik kimeekiqis gsnykslevl vadlvnaqkd
    421 smgdessqts lqkeisteeq lrrlqeeklc kicmdrniai vfvpcghlvt ckqcaeavdk
    481 cpmcytvitf kqkifms
    • EQUIVALENTS
  • It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims:

Claims (21)

1-282. (canceled)
283. An immunogenic composition comprising one or more antigens or immunogenic fragments thereof;
wherein the one or more antigens are selected by a method comprising:
a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen;
b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads;
c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs;
d) determining whether one or more lymphocytes are activated by one or more tumor antigens presented by one or more APCs by assessing a level of expression and/or secretion of one or more immune mediators;
e) identifying each activating tumor antigen as (i) an antigen that stimulates the level of expression and/or secretion of one or more immune mediators, or (ii) an antigen that inhibits and/or suppresses the level of expression and/or secretion of one or more immune mediators; and
f) selecting from among the identified tumor antigens (i) one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, (ii) one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, (iii) one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (iv) one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer.
284. The immunogenic composition of claim 283, wherein the immunogenic composition does not comprise: (iii) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (iv) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, or immunogenic fragments thereof.
285. The immunogenic composition of claim 283, wherein the immunogenic composition comprises: (i) one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, or immunogenic fragments thereof.
286. A method of manufacturing an immunogenic composition, the method comprising:
combining one or more selected antigens or immunogenic fragments thereof and a carrier;
wherein the one or more selected antigens are selected by a method comprising:
a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen;
b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads;
c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs;
d) determining whether one or more lymphocytes are activated by one or more tumor antigens presented by one or more APCs by assessing a level of expression and/or secretion of one or more immune mediators;
e) identifying each activating tumor antigen as (i) an antigen that stimulates the level of expression and/or secretion of one or more immune mediators, or (ii) an antigen that inhibits and/or suppresses the level of expression and/or secretion of one or more immune mediators; and
f) selecting from among the identified tumor antigens (i) one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, (ii) one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, (iii) one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (iv) one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer.
287. The method of claim 286, wherein the APCs are human APCs isolated from the subject.
288. The method of claim 286, wherein the APCs and lymphocytes are isolated from peripheral blood.
289. The method of claim 286, wherein the lymphocytes are derived from a cancer or tumor.
290. The method of claim 286, wherein lymphocyte activation is determined by (i) assessing a level of one or more expressed or secreted immune mediators that is at least about 20% higher or lower than a control level; (ii) assessing a level of one or more expressed or secreted immune mediators that is at least two standard deviations greater or lower than the mean of a control level; and/or (iii) assessing a level of one or more expressed or secreted immune mediators that is at least 2 median absolute deviations (MADs) greater or lower than a median response level to a control.
291. The method of claim 286, wherein lymphocyte non-responsiveness is determined by (i) assessing a level of one or more expressed or secreted immune mediators that is within about 20% of a control level; (ii) assessing a level of one or more expressed or secreted immune mediators that is less than one standard deviation higher or lower than the mean of a control level; and/or (iii) assessing a level of one or more expressed or secreted immune mediators that is less than one median absolute deviation (MAD) higher or lower than a median response level to a control.
292. The method of claim 286, wherein the APCs are provided in an array, and wherein the APCs in each location of the array are contacted with a set of bacterial cells, each set comprising a different tumor antigen.
293. A method of identifying a subject as a candidate for cancer therapy, the method comprising:
1) obtaining a subject response profile, wherein the subject response profile is generated by:
a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen;
b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from a subject, wherein the APCs internalize the bacterial cells or beads;
c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs;
d) determining whether one or more lymphocytes are activated by one or more tumor antigens presented by one or more APCs by assessing a level of expression and/or secretion of one or more immune mediators;
e) identifying each activating tumor antigen as (i) an antigen that stimulates the level of expression and/or secretion of one or more immune mediators, or (ii) an antigen that inhibits and/or suppresses the level of expression and/or secretion of one or more immune mediators, to obtain or generate a subject response profile; and
2) determining, based on one or more characteristics of the subject response profile, whether the subject is a candidate subject for initiation, continuation, modification, discontinuation or non-initiation of a cancer therapy.
294. The method of claim 293, wherein the subject response profile comprises a representation of the level of expression and/or secretion of the one or more immune mediators associated with the plurality of tumor antigens.
295. The method of claim 293, wherein the one or more characteristics of the subject response profile include:
(i) number of activating tumor antigens identified;
(ii) number of antigens that stimulate the level of expression and/or secretion of one or more immune mediators; and/or
(iii) number of antigens that inhibit and/or suppress the level of expression and/or secretion of one or more immune mediators.
296. The method of claim 293, wherein the subject response profile is in the form of a report in paper and/or electronic form.
297. The method of claim 293, wherein the report also contains information on prognosis, resistance, or potential or suggested therapeutic options; information on the likely effectiveness of a therapeutic option; the acceptability of a therapeutic option; the advisability of applying the therapeutic option to a cancer patient; and/or a recommendation on the administration of a cancer therapy (e.g., the administration of a pre-selected dosage or in a preselected treatment regimen e.g., in combination with one or more alternative cancer therapies).
298. The method of claim 293, wherein the subject suffers from a tumor.
299. The method of claim 298, wherein the tumor is or comprises a solid tumor selected from a breast carcinoma, a squamous cell carcinoma, a colon cancer, a head and neck cancer, ovarian cancer, a lung cancer, mesothelioma, a genitourinary cancer, a rectal cancer, a gastric cancer, or an esophageal cancer.
300. The method of claim 293, wherein the cancer therapy comprises an immunogenic composition comprising: (i) one or more antigens that increase a level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, and/or (ii) one or more tumor antigens that inhibit and/or suppress a level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, or immunogenic fragments thereof.
301. The method of claim 293, wherein the cancer therapy further comprises at least one immune checkpoint inhibitor.
302. The method of claim 293, wherein the immunogenic composition does not comprise (iii) one or more tumor antigens that increase level of expression and/or secretion of one or more immune mediators associated with at least one deleterious and/or non-beneficial response to cancer, and/or (iv) one or more tumor antigens that inhibit and/or suppress level of expression and/or secretion of one or more immune mediators associated with at least one beneficial response to cancer, or immunogenic fragments thereof.
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