[go: up one dir, main page]

WO2019204663A1 - Formulations peptidiques et utilisations associées - Google Patents

Formulations peptidiques et utilisations associées Download PDF

Info

Publication number
WO2019204663A1
WO2019204663A1 PCT/US2019/028204 US2019028204W WO2019204663A1 WO 2019204663 A1 WO2019204663 A1 WO 2019204663A1 US 2019028204 W US2019028204 W US 2019028204W WO 2019204663 A1 WO2019204663 A1 WO 2019204663A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
neo
antigenic peptides
concentration
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2019/028204
Other languages
English (en)
Inventor
Zhengxin Dong
Robyn Jessica EISERT
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.)
Biontech US Inc
Original Assignee
Neon Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Neon Therapeutics Inc filed Critical Neon Therapeutics Inc
Publication of WO2019204663A1 publication Critical patent/WO2019204663A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells

Definitions

  • neoplasia vaccines that contain tumor-specific and/or patient-specific neo-antigens. These neo-antigens are ideal for enhancing T cell responses to a particular tumor type, thus overcoming some of the disadvantages of vaccines containing shared tumor antigens.
  • many neo-antigenic peptides are poorly soluble in many pharmaceutically acceptable carriers, which prevent their use in vaccines or other pharmaceutical formulations. Poor solubility can lead to poor adsorption after administration, limit the shelf life of a formulation and reduce its efficacy after reconstitution.
  • the present disclosure addresses the development of formulations for improving the solubility of neo- antigenic peptides and enables the use of previously insoluble neo-antigenic peptides to be used in a pharmaceutical formulation.
  • a pharmaceutical composition comprising: one or more neo-antigenic peptides or a pharmaceutically acceptable salt thereof; a pH modifier present at a concentration of less than 1.0 mM; and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a vaccine composition.
  • the pharmaceutical composition is aqueous.
  • the pharmaceutical composition is lyophilizable.
  • At least one of the one or more neo-antigenic peptides is bounded by pi >5 and HYDRO >-6.
  • “is bounded by” is equivalent to“has a”.
  • a peptide that is bounded by pl>5 and HYDRO ⁇ -6 is equivalent to a peptide that has a pl>5 and HYDRO ⁇ -6.
  • at least one of the one or more neo-antigenic peptides is bounded by pl>8 and HYDRO >-8.
  • At least one of the one or more neo-antigenic peptides is bounded by pl ⁇ 5 and HYDRO >-5. In some embodiments, at least one of the one or more neo-antigenic peptides is bounded by pl>9 and HYDRO ⁇ -8. In some embodiments, at least one of the one or more neo-antigenic peptides is bounded by pi >7 and a HYDRO value of >-5.5. In some embodiments, at least one of the one or more neo- antigenic peptides is bounded by pl>0 and HYDRCK-8, pl>0 and HYDRO >-4, or pl>4.3 and - 4 ⁇ HYDRO>-8.
  • At least one of the one or more neo-antigenic peptides is bounded by pl>0 and HYDRO>-4, or pl>4.3 and HYDRO ⁇ -4. In some embodiments, at least one of the one or more neo-antigenic peptides is bounded by pl>0 and HYDRO>-4, or pl>4.3 and -4 ⁇ HYDRO>-9. In some embodiments, at least one of the one or more neo-antigenic peptides is bounded by 5 ⁇ pi >12 and -4 ⁇ HYDRO>-9. In some embodiments, at least one of the one or more neo-antigenic peptides is bounded by pi ⁇ 4.3 and -4 ⁇ HYDRO>-8.
  • At least one of the one or more neo-antigenic peptides of the pharmaceutical composition has 5 ⁇ _p I >12 and -6 ⁇ HYDRO>-9.
  • At least one of the one or more neo-antigenic peptides of the pharmaceutical composition has 7.5 ⁇ pl >12 and -4 ⁇ HYDRO>-9.
  • At least one of the one or more neo-antigenic peptides of the pharmaceutical composition has a pi of 5 to 6.5 or 7.5 to 12 and -4 ⁇ HYDRO>-9.
  • At least one of the one or more neo-antigenic peptides of the pharmaceutical composition has 5 ⁇ pl >12 and has a HYDRO of -4 to -4.9 or -5.8 to -9.
  • At least one of the one or more neo-antigenic peptides of the pharmaceutical composition has a pi of 5 to 6.5 or 7.5 to 12 and has a HYDRO of -4 to -4.9 or - 5.8 to -9.
  • the pharmaceutical composition comprises at least one neo- antigenic peptide. In some embodiments, the pharmaceutical composition comprises at least two neo-antigenic peptides. In some embodiments, the pharmaceutical composition comprises at least three neo-antigenic peptides. In some embodiments, the pharmaceutical composition comprises at least four neo-antigenic peptides. In some embodiments, the pharmaceutical composition comprises at least five neo-antigenic peptides. In some embodiments, the pharmaceutical composition comprises up to 40 neo-antigenic peptides. In some embodiments, each of the one or more neo-antigenic peptides is soluble in the pharmaceutical composition.
  • each of the one or more neo-antigenic peptide comprises from 6 to 50 amino acids, 6 to 45 amino acids, 6 to 40 amino acids, 6 to 35 amino acids, 6 to 30 amino acids, 6 to 25 amino acids, 6 to 20 amino acids, 8 to 50 amino acids, 8 to 45 amino acids, 8 to 40 amino acids, 8 to 35 amino acids, 8 to 30 amino acids, 8 to 25 amino acids, 8 to 20 amino acids, 14 to 50 amino acids, 14 to 45 amino acids, 14 to 40 amino acids, 14 to 35 amino acids, 14 to 30 amino acids, 14 to 27 amino acids, 14 to 25 amino acids, or 14 to 20 amino acids.
  • at least one of the one or more neo-antigenic peptides comprises from 14 to 30 amino acids.
  • at least one of the one or more neo-antigenic peptides is less than 15 amino acids or less in length, from 7 to 11 amino acids in length, or from 8 to 10 amino acids in length.
  • At least one of the one or more neo-antigenic peptides is 40 amino acids or less in length, from 6 to 25 amino acids in length, from 14 to 30 amino acids in length, or from 14 to 27 amino acids in length.
  • the pH modifier is a base. In some embodiments, the pH modifier is a conjugate base of a weak acid. In some embodiments, the pH modifier is a pharmaceutically acceptable salt. In some embodiments, the pH modifier is a dicarboxylate or tricarboxylate salt. In some embodiments, the pH modifier is citric acid and/or a citrate salt. In some embodiments, the citrate salt is disodium citrate and/or trisodium citrate. In some embodiments, the pH modifier is succinic acid and/or a succinate salt. In some embodiments, the succinate salt is disodium succinate and/or monosodium succinate. In some embodiments, the disodium succinate is disodium succinate hexahydrate.
  • the pH modifier is present at a concentration of 0.75 mM or less. In some embodiments, the pH modifier is present at a concentration of 0.50 mM or less. In some embodiments, the pH modifier is present at a concentration of 0.25 mM or less. In some embodiments, the pH modifier is present at a concentration of from 0.25 mM to 1.0 mM. In some embodiments, the pH modifier is present at a concentration of from 0.50 mM to 1.0 mM. In some embodiments, the pH modifier is present at a concentration of from 0.75 mM to 1.0 mM. In some embodiments, the pH modifier is present at a concentration of from 0.50 mM to 0.75 mM.
  • the pharmaceutically acceptable carrier comprises a liquid. In some embodiments, the pharmaceutically acceptable carrier comprises water. In some embodiments, the pharmaceutically acceptable carrier comprises a sugar. In some embodiments, the sugar comprises dextrose. In some embodiments, the dextrose is present at a concentration of 5% w/v. In some embodiments, the sugar comprises trehalose. In some embodiments, the sugar comprises sucrose. In some embodiments, the pharmaceutically acceptable carrier comprises dimethyl sulfoxide (DMSO). In some embodiments, the DMSO is present at a concentration from 0.1% to 10%, 0.5% to 5%, or 1% to 3%.
  • DMSO dimethyl sulfoxide
  • the pharmaceutical composition further comprises an immunomodulator or adjuvant.
  • the immunomodulator or adjuvant is selected from the group consisting of poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonists, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryllipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGA microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles,
  • the immunomodulator or adjuvant comprises poly-ICLC.
  • a ratio of poly-ICLC to neo-antigenic peptides in the pharmaceutical composition is from 2: 1 to 1 :10 v:v. In some embodiments, the ratio of poly-ICLC to neo-antigenic peptides in the pharmaceutical composition is about 1 : 1, 1 :2, 1 :3, 1 :4 or 1 :5 v:v.
  • a pharmaceutical composition comprising: one or more neo-antigenic peptides; dimethylsulfoxide present at a concentration of at least 0.5%, 1%, 2%, 3% or more; a sugar present at an concentration of at least 1% w/v, 2% w/v, 3% w/v, 4% w/v, 5% w/v or more; and succinic acid or a succinate salt present at a concentration of less than 1.0 mM.
  • a pharmaceutical composition comprising: one or more neo-antigenic peptides; 1-10% dimethylsulfoxide; 1-10% w/v of a sugar in water; and lower than 1.0 mM succinic acid or a succinate salt.
  • a pharmaceutical composition comprising: one or more neo-antigenic peptides; 1-10% dimethylsulfoxide; 1-10% w/v of a sugar in water; and lower than 1.0 mM succinic acid or a succinate salt; and poly Lpoly C.
  • the poly Lpoly C comprises poly-L-Lysine; In some embodiments, the poly Lpoly C comprises sodium carboxymethylcellulose. In some embodiments, the pharmaceutical composition further comprises sodium chloride.
  • a pharmaceutical composition comprising: one to five neo-antigenic peptides, 1-4% v/v dimethylsulfoxide, 3-5% dextrose, succinic acid or a succinate salt present at a concentration of less than 1.0 mM.
  • a pharmaceutical composition comprising: one to five neo-antigenic peptides, 1-4% dimethylsulfoxide, 3-5% dextrose, succinic acid or a succinate salt present at a concentration of less than 1.0 mM, 0.4-0.6 mg/ml poly I: poly C, 0.3-0.5 mg/ml poly-L-Lysine, 0.5-2 mg/ml sodium carboxymethylcellulose, and 0.1-0.5% sodium chloride.
  • each of the one or more neo-antigenic peptides are present at a concentration at least 1 pg/mL, at least 10 pg/mL, at least 25 pg/mL, at least 50 pg/mL, or at least 100 pg/mL. In some embodiments, each of the one or more neo-antigenic peptides are present at a concentration at most 5000 pg/mL, at most 2500 pg/mL, at most 1000 pg/mL, at most 750 pg/mL, at most 500 pg/mL, at most 400 pg/mL, or at most 300 pg/mL.
  • each of the one or more neo-antigenic peptides are present at a concentration of from 10 pg/mL to 5000 pg/mL, 10 pg/mL to 4000 pg/mL, 10 pg/mL to 3000 pg/mL, 10 pg/mL to 2000 pg/mL, 10 pg/mL to 1000 pg/mL, 25 pg/mL to 500 pg/mL, or 50 pg/mL to 300 pg/mL.
  • a higher percentage of neo-antigenic peptides are soluble in the pharmaceutical composition compared to a pharmaceutical composition comprising the pH modifier at a concentration of 1.0 mM or higher. In some embodiments, a higher percentage of the one or more neo-antigenic peptides are soluble in the pharmaceutical composition compared to a pharmaceutical composition comprising wherein the pH modifier is present at a concentration of 1.0 mM or higher.
  • a vaccine comprising a pharmaceutical composition provided herein.
  • the vaccine is a neoplasia vaccine.
  • an immunogenic composition comprising a pharmaceutical composition provided herein.
  • a vaccination or immunization kit comprising: a lyophilized composition comprising one or more neo-antigenic peptides; a pH modifier; and instructions for combining (a) and (b) into a solution, wherein the solution comprises the pH modifier at a concentration of less than 1.0 mM.
  • the kit further comprises a viral vector.
  • the kit further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier is water.
  • a vaccination or immunization kit comprising: a composition comprising one or more lyophilized neo-antigenic peptides or a pharmaceutically acceptable salt thereof; a pH modifier; and instructions for combining (a) and (b) into a solution, wherein the solution comprises the pH modifier at a concentration of less than 1.0 mM.
  • the vaccination or immunization kit further comprises an adjuvant. In some embodiments, the vaccination or immunization kit further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is water.
  • a method of preparing a pharmaceutical composition comprising combining one or more lyophilized neo-antigenic peptides with a pH modifier and a pharmaceutically acceptable carrier to form a peptide solution, wherein the pH modifier is present at a concentration of less than 1.0 mM.
  • the method further comprises filtering the peptide solution.
  • a method of preparing a pharmaceutical composition comprising combining at one or more neo-antigenic peptides with a pH modifier and a pharmaceutically acceptable carrier to form a peptide solution, wherein the pH modifier is present at a concentration of less than 1.0 mM.
  • the method further comprises filtering the peptide solution.
  • the one or more neo-antigenic peptides are lyophilized before combining.
  • a method of preparing a vaccine comprising combining a pharmaceutical composition provided herein with an immunomodulator or adjuvant.
  • a method of treating a subject with a condition or disease comprising administering a pharmaceutical composition provided herein to the subject, thereby treating the condition or disease.
  • the method further comprises administering another therapeutic agent to the subject. In some embodiments, the method further comprises administering a second pharmaceutical composition to the subject, wherein the second pharmaceutical composition is a pharmaceutical composition provided herein. In some embodiments, the method further comprises administering a third pharmaceutical composition to the subject, wherein the third pharmaceutical composition is a pharmaceutical composition provided herein. In some embodiments, the method further comprises administering a fourth pharmaceutical composition to the subject, wherein the fourth pharmaceutical composition is a pharmaceutical composition provided herein. In some embodiments, the method further comprises administering at least a fifth, sixth, seventh, or eighth pharmaceutical composition to the subject, wherein the at least fifth, sixth, seventh, or eighth pharmaceutical composition is a pharmaceutical composition provided herein.
  • the condition or disease is a neoplasia.
  • the neoplasia is a cancer.
  • an aqueous neo-antigenic peptide solution comprising: one or more neo-antigenic peptides; and succinic acid or a succinate salt present at a concentration of less than 1.0 mM.
  • aqueous neo-antigenic peptide solution comprising combining one or more neo-antigenic peptides with succinic acid or a succinate salt and a liquid carrier, wherein the succinic acid or succinate salt is present at a concentration of less than 1.0 mM.
  • a method of increasing the solubility of one or more neo-antigenic peptides comprising adjusting a concentration of a pH modifier present in a pharmaceutical composition comprising one or more neo-antigenic peptides, the pH modifier, and a pharmaceutically acceptable carrier; wherein adjusting comprises lowering the concentration of a pH modifier to a concentration of less than 1.0 mM.
  • FIG. 1 shows that all twenty of the peptides that were soluble in the pharmaceutical compositions with a succinate concentration of 5 mM remain soluble when succinate concentration is reduced to 0.75 mM, 0.5 mM, and 0.25 mM.
  • FIG. 2 shows that fourteen out of twenty peptides that were insoluble in the pharmaceutical compositions with a succinate concentration of 5 mM became soluble at lower succinate concentrations. Among the fourteen peptides that became soluble at the lower succinate concentrations, three were only soluble at succinate concentrations of about 0.25 mM.
  • FIG. 3 shows that pharmaceutical compositions comprising three different succinate concentrations (5 mM, 0.5 mM, and 0.25 mM) containing a combination of peptides and poly- ICLC in a 3 : 1 ratio resulted in cloudy suspensions comparable to poly-ICLC alone, and did not result in precipitation of nucleic acids of poly-ICLC.
  • FIG. 4 shows a chart plotting the HYDRO and pi values of the sets of neo-antigenic peptides shown in FIG. 2 and FIG. 3 and described herein.
  • FIG. 5 shows a chart plotting the hydrophobicity of the peptides calculated based on Kyte-Doolittle method and their pi values for the sets of neo-antigenic peptides shown in FIG. 2 and FIG. 3 and described herein.
  • the term“about” or“approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.
  • “about” can mean within 1 or more than 1 standard deviation, per the practice in the art.
  • “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value.
  • Ranges provided herein are meant to include all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.
  • analog is to be construed to mean a molecule that is not identical, but has analogous functional or structural features.
  • a tumor specific neo-antigen polypeptide analog retains the biological activity of a corresponding naturally-occurring tumor specific neo-antigen polypeptide, while having certain biochemical modifications that may enhance the analog's function relative to a naturally-occurring polypeptide.
  • biochemical modifications could increase the analog's protease resistance, membrane permeability, or half- life, without altering, for example, ligand binding.
  • An analog may include a non-standard or unnatural amino acid.
  • neo-antigen and“neoantigen” are used interchangeably.
  • the term“neo- antigenic” and“neoantigenic” are used interchangeably.
  • the term“neo-antigen,”“neoantigen,” “neo-antigenic,” or“neoantigenic” can refer to antigens that are not encoded in a normal, non- mutated host genome.
  • a neo-antigen can relate to an antigen including one or more amino acid modifications compared to the parental antigen.
  • a neo-antigen may be a tumor- associated neo-antigen, wherein the term“tumor-associated neo-antigen” can include a peptide or protein including amino acid modifications due to tumor-specific mutations.
  • a neo-antigen represents either oncogenic viral proteins or abnormal proteins that arise as a consequence of somatic mutations.
  • a neo-antigen can arise by the disruption of cellular mechanisms through the activity of viral proteins.
  • Another example can be an exposure to a carcinogenic compound, which in some cases can lead to a somatic mutation. This somatic mutation can ultimately lead to the formation of a tumor/cancer.
  • neoplasia is to be construed to mean any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both.
  • cancer is an example of a neoplasia.
  • cancers include, without limitation, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non- Hodgkin’s disease), Waldenstrom’s macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangio
  • the term “vaccine” can relate to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, in particular, a cellular immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell.
  • a vaccine may be used for the prevention or treatment of a disease.
  • neoplasia vaccine is meant to refer to a combination of neoplasia/tumor specific neoantigens, for example at least two, at least three, at least four, at least five, or more neo-antigenic peptides.
  • A“vaccine” is to be understood as meaning a composition for generating immunity for the prophylaxis and/or treatment of diseases (e.g., neoplasia/tumor). Accordingly, vaccines are medicaments which comprise antigens and are intended to be used in humans or animals for generating specific defense and protective substance by vaccination.
  • A“neoplasia vaccine composition” can include a pharmaceutically acceptable excipient, carrier or diluent.
  • the term“pharmaceutically acceptable” can refer to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, including humans.
  • A“pharmaceutically acceptable carrier, excipient, or diluent” can refer to a carrier, excipient, or diluent that can be administered to a subject together with an active ingredient(s), such as small molecule chemical compound, antibody, nucleic acid molecule, peptide, polypeptide, and fragments thereof, and that does not destroy the pharmacological activity of the active ingredient(s).
  • an active ingredient(s) such as small molecule chemical compound, antibody, nucleic acid molecule, peptide, polypeptide, and fragments thereof, and that does not destroy the pharmacological activity of the active ingredient(s).
  • A“pharmaceutically acceptable salt” of a neo-antigenic peptide, or a combination of neo- antigenic peptides can refer to an acid or base salt that is generally considered in the art to be suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication.
  • Such salts include mineral and organic acid salts of basic residues such as amines, as well as alkali or organic salts of acidic residues such as carboxylic acids.
  • Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfuric, sulfamic, sulfanilic, formic, toluenesulfonic, methanesulfonic, benzene sulfonic, ethane disulfonic, 2- hydroxyethylsulfonic, nitric, benzoic, 2-acetoxybenzoic, citric, tartaric, lactic, stearic, salicylic, glutamic, ascorbic, pamoic, succinic, fumaric, maleic, propionic, hydroxymaleic, hydroiodic, phenylacetic, alkanoic such as acetic, HOOC-(CH 2 ) n -COOH where n is 0-4, and the like.
  • acids such as hydrochloric, phosphoric, hydrobromic, malic, glycolic, fumaric, sulfur
  • pharmaceutically acceptable cations include, but are not limited to sodium, potassium, calcium, aluminum, lithium, and ammonium.
  • a “pharmaceutically acceptable salt” of a neo-antigenic peptide can also refer to those listed by Remington’s Pharmaceutical Sciences, l7th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985).
  • a pharmaceutically acceptable acid or base salt can be synthesized from a parent compound that contains a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in an appropriate solvent.
  • polypeptide or“peptide” can refer to a polypeptide that has been separated from components that naturally accompany it. Unless otherwise specified, the term “polypeptide” or“peptide” encompasses lyophilized peptide and the pharmaceutically acceptable salts of the peptide. Typically, the peptide provided herein has a purity of at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Alternatively, the peptide has a purity of at least 75%, at least 90%, or at least 99%, by weight.
  • a peptide may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a peptide; or by chemical synthesis.
  • the purity of a peptide can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, and HPLC analysis.
  • subject can refer to an animal which is the object of treatment, observation or experiment.
  • a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, bovine, equine, canine, ovine, or feline.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” and the like refer to reducing the probability of developing a disease or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease or condition.
  • “treat,”“treated,”“treating,”“treatment,” and the like are meant to refer to reducing or ameliorating a disorder and/or symptoms associated therewith (e.g., a neoplasia or tumor).“Treating” includes the concepts of“alleviating”, which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to a cancer and/or the side effects associated with cancer therapy.
  • treating also encompasses the concept of“managing” which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.
  • reduces is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.
  • “therapeutic effect” refers to some extent of relief of one or more of the symptoms of a disorder (e.g., a neoplasia or tumor) or its associated pathology.
  • “Therapeutically effective amount” as used herein refers to an amount of an agent which is effective, upon single or multiple dose administration to the cell or subject, in prolonging the survivability of the patient with such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying, and the like beyond that expected in the absence of such treatment.“Therapeutically effective amount” is intended to qualify the amount required to achieve a therapeutic effect.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the “therapeutically effective amount” (e.g., ED50) of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds employed in a pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the pharmaceutical compositions typically should provide a dosage of from about 0.0001 mg to about 200 mg of compound per kilogram of body weight per day.
  • dosages for systemic administration to a human patient can range from 0.01-10 pg/kg, 20-80 mg/kg, 5-50 pg/kg, 75-150 pg/kg, 100-500 mg/kg, 250-750 pg/kg, 500-1000 pg/kg, 1-10 mg/kg, 5-50 mg/kg, 25-75 mg/kg, 50-100 mg/kg, 1.00-250 mg/kg, 50-100 mg/kg, 250-500 mg/kg, 500-750 mg/kg, 750-1000 mg/kg, 1000-1500 mg/kg, 1500-2000 mg/kg, 5 mg/kg, 20 mg/kg, 50 mg/kg, 1.00 mg/kg, of 200 mg/kg.
  • Pharmaceutical dosage unit forms are prepared to provide from about 0.001 mg to about 5000 mg, for example from about 100 to about 2500 mg of the compound or a combination of essential ingredients per
  • the present disclosure relates to vaccines and methods for the treatment of neoplasia, and more particularly tumors, through the administration of a therapeutically effective amount of a pharmaceutical composition (e.g., a cancer vaccine) comprising a plurality of neoplasia/tumor specific neo-antigens to a subject (e.g., a mammal such as a human).
  • a pharmaceutical composition e.g., a cancer vaccine
  • a subject e.g., a mammal such as a human.
  • whole genome/exome sequencing may be used to identify all, or nearly all, mutated neo-antigens that are uniquely present in a neoplasia/tumor of an individual patient, and that this collection of mutated neo-antigens may be analyzed to identify a specific, optimized subset of neo-antigens for use as a personalized cancer vaccine or immunogenic composition for the treatment of the patient's neoplasia/tumor.
  • a population of neoplasia/tumor specific neo-antigens may be identified by sequencing the neoplasia/tumor and normal DNA of each patient to identify tumor-specific mutations, and the patient's human leukocyte antigen (HLA) allotype can be identified.
  • the population of neoplasia/tumor specific neo-antigens and their cognate native antigens may then be subject to bioinformatic analysis using validated algorithms to predict which tumor-specific mutations create epitopes that could bind to the patient's HLA allotype.
  • a plurality of peptides corresponding to a subset of these mutations may be designed and synthesized for each patient, and combined together for use as a cancer vaccine or immunogenic composition in immunizing the patient.
  • the neo-antigens peptides may be combined with an adjuvant (e.g., poly-ICLC) or another anti -neoplastic agent.
  • an adjuvant e.g., poly-ICLC
  • these neo-antigens are expected to bypass central thymic tolerance (thus allowing stronger anti-tumor T cell response), while reducing the potential for autoimmunity (e.g., by avoiding targeting of normal self-antigens).
  • the immune system can be classified into two functional subsystems: the innate and the acquired immune system.
  • the innate immune system is the first line of defense against infections, and most potential pathogens are rapidly neutralized by this system before they can cause, for example, a noticeable infection.
  • the acquired immune system reacts to molecular structures, referred to as antigens, of an intruding organism.
  • humoral immune reaction antibodies secreted by B cells into the bodily fluids bind to pathogen- derived antigens, leading to the elimination of the pathogen through a variety of mechanisms, e.g. complement-mediated lysis.
  • T-cells capable of destroying other cells are activated. For example, if proteins associated with a disease are present in a cell, they are fragmented proteolytically to peptides within the cell. Specific cell proteins then attach themselves to the antigen or peptide formed in this manner and transport them to the surface of the cell. Once there they are presented to the molecular defense mechanisms, in particular T-cells, of the body. Cytotoxic T cells recognize these antigens and kill cells that harbor the antigens.
  • MHC proteins that transport and present peptides on the cell surface are referred to as proteins of the major histocompatibility complex (MHC).
  • MHC proteins are classified into two types, referred to as MHC class I and MHC class II. While the structures of the proteins of the two MHC classes are very similar, they have very different functions. Proteins of MHC class I are present on the surface of almost all cells of the body, including most tumor cells. MHC class I proteins are loaded with antigens that typically originate from endogenous proteins or from pathogens present inside cells, and are then presented to naive or cytotoxic T-lymphocytes (CTLs).
  • CTLs cytotoxic T-lymphocytes
  • MHC class II proteins are present on dendritic cells, B- lymphocytes, macrophages and other antigen-presenting cells. They mainly present peptides, which are processed from external antigen sources (e.g., outside of the cells, to T-helper (Th) cells). Most peptides bound by the MHC class I proteins originate from cytoplasmic proteins produced in the healthy host cells of an organism itself, and do not normally stimulate an immune response. Accordingly, cytotoxic T-lymphocytes that recognize such self-peptide-presenting MHC molecules of class I are deleted in the thymus (central tolerance) or, after their release from the thymus, are deleted or inactivated (e.g., tolerized (peripheral tolerance)).
  • MHC molecules are capable of stimulating an immune reaction when they present peptides to non-tolerized T-lymphocytes.
  • Cytotoxic T-lymphocytes have both T-cell receptors (TCR) and CD8 molecules on their surface.
  • T-Cell receptors are capable of recognizing and binding peptides complexed with the molecules of MHC class I.
  • Each cytotoxic T-lymphocyte expresses a unique T-cell receptor which is capable of binding specific MHC/peptide complexes.
  • the peptide antigens attach themselves to molecules of MHC class I by competitive affinity binding within the endoplasmic reticulum, before they are presented on the cell surface.
  • affinity of an individual peptide antigen is directly linked to its amino acid sequence and the presence of specific binding motifs in defined positions within the amino acid sequence. Therefore, if the sequence of such a peptide is known, it may be possible to manipulate the immune system against diseased cells using, for example, peptide vaccines.
  • Tumor neo-antigens which arise as a result of genetic change (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) within malignant cells, represent the most tumor-specific class of antigens.
  • neo-antigens have rarely been used in cancer vaccine or immunogenic compositions due to technical challenges in identifying them, selecting optimized neo-antigens, and producing neo-antigens for use in a vaccine or immunogenic composition.
  • translating sequencing information into a therapeutic vaccine may include:
  • the mutated epitopes of a cancer patient are determined.
  • mutated epitopes are determined by sequencing the genome and/or exome of tumor tissue and healthy tissue from a cancer patient using next generation sequencing technologies.
  • genes that are selected based on their frequency of mutation and ability to act as a neo-antigen are sequenced using next-generation sequencing technology.
  • Next-generation sequencing can apply to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChIP-sequencing), and epigenome characterization (de Magalhaes JP, Finch CE, Janssens G (2010).“Next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions”. Ageing Research Reviews 9 (3): 315-323; Hall N (May 2007).
  • NeoORFs are particularly valuable as immunogens because the entirety of their sequence is completely novel to the immune system and as such are analogous to a viral or bacterial foreign antigen.
  • neoORFs (1) are highly specific to the tumor (i.e.
  • mutated proteins are valuable targets for a host's immune response to the tumor as, unlike native proteins, they are not subject to the immune-dampening effects of self-tolerance. Therefore, mutated proteins are more likely to be immunogenic and are also more specific to the tumor cells compared to the normal cells of a patient.
  • An alternative method for identifying tumor specific neo-antigens is through 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 neo-antigens.
  • MSn multidimensional MS techniques
  • MS/MS tandem mass spectrometry
  • Such proteomic approaches permit rapid, highly automated analysis (see, e.g., K. Gevaert and J. Vandekerckhove, Electrophoresis 21 : 1145-1154 (2000)). It is further contemplated that high-throughput methods for the de novo sequencing of unknown proteins may be used to analyze the proteome of a patient's tumor in order to identify expressed neo antigens.
  • meta shotgun protein sequencing may be used to identify expressed neo antigens (see e.g., Guthals et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(10): 1084-96).
  • Tumor specific neoantigens may also be identified using MHC multimers to identify neo antigen-specific T-cell responses.
  • MHC multimers to identify neo antigen-specific T-cell responses.
  • high-throughput analysis of neo-antigen-specific T-cell responses in patient samples may be performed using MHC tetramer-based screening techniques (see e.g., Hombrink et al. (2011) High-Throughput Identification of mutant epitopes to an effective vaccine or immunogenic composition.
  • Peptides can be readily synthesized chemically and purified easily through the utilization of reagents free of contaminating bacteria or animal substances. The small size allows a clear focus on the mutated region of the protein and also reduces irrelevant antigenic competition from other components (unmutated protein or viral vector antigens).
  • the drug formulation is a multi-epitope vaccine or immunogenic composition of long peptides.
  • Such“long” peptides undergo efficient internalization, processing and cross-presentation in professional antigen-presenting cells such as dendritic cells, and have been shown to induce CTLs in humans (Melief and van der Burg, Immunotherapy of established (pre) malignant disease by synthetic long peptide vaccines Nature Rev Cancer 8:351 (2008)).
  • at least 1 neo-antigenic peptide is prepared for immunization.
  • at least 2 neo-antigenic peptides are prepared for immunization.
  • At least 3 neo-antigenic peptides are prepared for immunization. In some embodiments, at least 4 neo-antigenic peptides are prepared for immunization. In some embodiments, at least 5 neo-antigenic peptides are prepared for immunization. In some embodiments, 20 or more peptides are prepared for immunization. In some embodiments, up to 40 neo-antigenic peptides are prepared for immunization.
  • the neoantigenic peptide ranges from about 5 to about 50 amino acids in length, about 5 to about 45 amino acids in length, about 5 to about 40 amino acids in length, about 5 to about 35 amino acids in length, about 5 to about 30 amino acids in length, about 5 to about 25 amino acids in length, about 5 to about 20 amino acids in length, about 10 to about 50 amino acids in length, about 10 to about 45 amino acids in length, about 10 to about 40 amino acids in length, about 10 to about 35 amino acids in length, about 10 to about 30 amino acids in length, about 10 to about 25 amino acids in length, about 10 to about 20 amino acids in length, about 15 to about 50 amino acids in length, about 15 to about 45 amino acids in length, about 15 to about 40 amino acids in length, about 15 to about 35 amino acids in length, about 15 to about 30 amino acids in length, about 15 to about 25 amino acids in length, about 15 to about 20 amino acids in length, about 14 to about 50 amino acids in length, about 14 to about 45 amino acids in length, about 14 to about 40 amino acids in length in length
  • peptides from about 14 to about 35 amino acids in length are synthesized. In some embodiments, peptides from about 14 to about 27 amino acids in length are synthesized. In some embodiments, the neoantigenic peptide ranges from about 20 to about 35 amino acids in length.
  • the present disclosure is based, at least in part, on the ability to present the immune system of the patient with a combination of tumor specific neoantigens.
  • tumor specific neoantigens may be produced either in vitro or in vivo.
  • Tumor specific neoantigens may be produced in vitro as peptides or polypeptides, which may then be formulated into a personalized neoplasia vaccine or immunogenic composition and administered to a subject.
  • tumor-specific neoantigens may be produced in vivo by introducing molecules (e.g., DNA, RNA, viral expression systems, and the like) that encode tumor specific neoantigens into a subject, whereupon the encoded tumor specific neoantigens are expressed.
  • the present disclosure is directed to pharmaceutical compositions with improved neo- antigenic peptide solubility.
  • the solubility of a peptide describes the peptide’s property of being soluble in a particular solution and in a particular condition. Solubility may also refer to the maximum quantity of a peptide that can dissolve in a certain quantity of a particular solvent and in a particular condition.
  • the solubility of a peptide may be dependent on the peptide’s structure, composition, and other characteristics, including but not limited to, the number of the residues in the peptide, the peptide’s size and molecular weight, the distribution and quantity of hydrophilic and hydrophobic residues, the distribution and quantity of negative and positive charges on the peptide, and the peptide’s isoelectric point (pi). For example, all other characteristics being equal, a peptide with shorter residues may have a higher solubility than a peptide with much longer residues.
  • the solubility of a peptide may also be dependent on the property of the solvent, such as its pH, hydrophobicity, hydrophilicity, polarity, boiling point, etc.
  • a peptide with consecutive hydrophobic residues may be less soluble in a hydrophilic solvent than a peptide without the consecutive hydrophobic residues.
  • Some of the hydrophobic and hydrophilic peptides that are insoluble in pure water may be soluble in polar organic solvents such as dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and acetonitrile; the relatively high polarity and dipole moments of those solvents may have made them more suitable to dissolve hydrophobic and/or charged peptides than pure water.
  • the solvent may be a mixture of a variety of different solvents (each individual solvent within the mixture, a“co-solvent”).
  • Each co-solvent may be soluble in the solvent mixture, and in a situation that a co-solvent is insoluble, then a stabilizer such as an emulsifier, a surfactant, a viscosity modifier, may be present in the solvent to improve its homogeneity.
  • a stabilizer such as an emulsifier, a surfactant, a viscosity modifier
  • the selection of a solvent may be limited by the application of the peptide.
  • a mixture of aqueous solution and a water-soluble organic solvent e.g., water/DMSO
  • Limitations in using organic solvents in injectable formulations may also include potential precipitation, pain, inflammation, and hemolysis upon injection.
  • the selection of a solvent may be further limited by the stability of the peptide in that solvent; the solvent should not react with or promote degradation of the peptide.
  • the solubility of a peptide in a particular solvent may be dependent on the temperature, pressure, the process and steps taken to dissolve the peptide, etc. Gentle sonication and heating are among the typical steps that can be taken to dissolve a peptide, and a peptide’s solubility may vary depending on the sequence, rate of addition, and how the co-solvents are being combined with the peptide.
  • the solubility of a peptide may be determined by experiment.
  • the peptide containing solutions are visually inspected-a clear solution without precipitations indicates that the peptide is soluble in that particular solvent or solvent mixture under a set of given conditions (e.g., fixed temperature and pressure).
  • the turbidity of the peptide containing solution can be used to determine if the peptide is soluble in a particular solvent or solvent mixture under a set of given conditions.
  • turbidity can be defined as a measure of the clarity of a fluid, liquid or solution and is an expression of the amount of light or radiation (e.g., visible light, Infrared radiation) that is scattered while passing through the fluid, liquid or solution.
  • the turbidity of a peptide containing solution can be qualitatively determined by visual inspection.
  • the turbidity can be measured using any instrument or technique suitable for measuring turbidity.
  • Such instruments and techniques include, but are not limited to, a turbidimeter or a turbidity sensor.
  • the turbidity of a peptide containing solution is compared to the turbidity of a reference solution whose composition is approximately identical but does not comprise a peptide. In some embodiments, when the turbidity values for the peptide containing and reference solutions are about equal the peptide is considered to be soluble in the solution under a particular set of conditions.
  • the turbidity value of the peptide solution is determined to be greater than that of the reference solution and when the difference in turbidity values has been determined to be statistically significant the peptide is considered to be partially soluble or insoluble in the solution.
  • the solubility of a peptide in an aqueous solution may also be predicted without experiment based on its amino acid sequence.
  • the solubility of a peptide in an aqueous solution may be predicted based on the peptide’s pi and hydrophobicity.
  • the solubility of a peptide may be predicted by using commonly available software programs. Peptides have certain pi and hydrophobicity characteristics may be more likely to be soluble in aqueous solutions than peptides with other pi and hydrophobicity characteristics.
  • a peptide’s isol electric point (pi) is the pH, at which the net charge of the peptide is zero.
  • a peptide possesses amino acid residues that may be positive, negative, or neutral, and the sum of the charges on each individual amino acid or termini is the net charge of the peptide.
  • the net charge (Z) of a peptide at a certain pH can be estimated by the following formula,
  • N is the number and pKai is the pKa value of the N-terminus and the side chains of Arginine (R), Lysine (K), and Histidine (H), and Nj is the number and pKa j is the pKa value of the C-terminus and the Aspartic acid (D), Glutamic Acid (E), Cysteine (C), and Tyrosine (Y) amino acid.
  • D Aspartic acid
  • E Glutamic Acid
  • C Cysteine
  • Y Tyrosine amino acid
  • the pi of a peptide may be estimated by using calculators, which are readily available online (e.g., that provided by pepcalc.com). At a pH below its pi, a peptide carries a net positive charge, and at a pH above its pi, a peptide carries a net negative charge. Thus, the pi of a peptide may affect its solubility. In some embodiments, the solubility of a peptide may be minimized in an aqueous solution by modifying the pH of the solution such that it corresponds to the pi of the peptide.
  • the hydrophobicity of a peptide may be determined by a variety of methods known to one of ordinary skill in the art.
  • the hydrophobicity of a peptide can be calculated using the Kyte-Doolittle method, which progressively evaluates the hydrophilicity and hydrophobicity of a peptide along its amino acid sequence (e.g., see Jack Kyte and Russell F. Doolittle, J. Mol. Biol. (1982) 157, 105-132).
  • the method utilizes a moving-segment approach to continuously determine the average hydropathy within a segment of predetermined length as it advances through the sequence. The consecutive scores are plotted from the amino to the carboxy terminus.
  • the midpoint line corresponds to the grand average of the hydropathy of the peptide and in some embodiments is referred to as the Kyte-Doolittle hydrophobicity value.
  • the known hydrophobicity scores of individual amino acids used in the Kyte-Doolittle method are provided in Table 2. [0089] Table 2 : Amino acid hydrophobicity scores used in Kyte-Doolittle method
  • the hydrophobicity of a peptide can be determined by calculating an index for the degree of hydrophobicity for each hydrophobic region of the peptide and then identifying the region with the highest degree of hydrophobicity. First, the different regions comprising one or more sequential hydrophobic amino acids in the peptide are identified. Thereafter the overall hydrophobicity or hydrophilicity score for each hydrophobic region is calculated by adding together the hydrophobicity /hydrophilicity values of each amino acid in the region. This calculation can be carried out using published values of hydrophobicity or hydrophilicity for each amino acid side chain. For example, the hydrophilicities for each of the 20 standard amino acids are provided in Table 3. Positive values correspond to hydrophilic amino acids and negative values correspond to hydrophobic amino acids. [0091] Table 3: Hydrophilicity values
  • a region of hydrophobic amino acids may be defined as one where all amino acids in the region have hydrophilicity values of less than 0.
  • the sum of the individual amino acid hydrophilicity values in a given hydrophobic sequence is the hydrophobicity index.
  • the contiguous sequence with the most negative hydrophobic index is the most hydrophobic.
  • the hydrophobicity index is referred to as HYDRO.
  • the hydrophobicity index of a peptide can be determined by identifying a region comprising one or more contiguous amino acids where each of the amino acids has a hydrophilicity value of less than 0, and wherein the region comprising the one or more amino acids has a total hydrophilicity value less than (e.g., more negative) any other region of the peptide comprising one or more amino acids.
  • the hydrophobicity index of a peptide having an amino acid sequence LEYVAFSQRFIPEL is -6.8
  • the hydrophobicity index of a peptide having an amino acid sequence L ARDIPP A VT GKWKL SDLRRY G A VP SG is -3.4.
  • the solubility of a peptide may be increased by the selection of a suitable solvent (including a solvent mixture).
  • a suitable solvent may be identified based on the physical properties of the peptide, including its isoelectric point (pi) and hydrophobicity, polarity and/or by experiment.
  • a suitable solvent may be a pure solvent or a solvent mixture, and for the latter, the composition of the co-solvents may also be adjusted to increase a peptide’s solubility.
  • the co-solvents are miscible and form a homogeneous solvent mixture.
  • one or more co-solvents may be immiscible with the solvent mixture and surfactants, emulsifiers, thickeners, or other stabilizers may be required to homogenize and stabilize the solvent mixture for extended storage.
  • the solubility of a peptide may be increased by changing the pH of a solvent; for example, adjusting the pH of a solvent away from the pi of the peptide through the addition of an acid or base.
  • the solubility of a peptide may be increased by sonication, heating, and/or adopting a suitable dissolution procedure.
  • the solubility of a peptide in a DMSO/water mixture may be improved by first dissolving the peptide in DMSO and then adding the water.
  • the solubility of a peptide may be increased through the addition of a complexing agent such as cyclic oligosaccharides like cyclodextrins or the addition of a chaotropic agent such as n-butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.
  • a complexing agent such as cyclic oligosaccharides like cyclodextrins
  • a chaotropic agent such as n-butanol, ethanol, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecy
  • the solubility of a peptide may be dependent on the ionic strength of a solvent.
  • the solubility may increase through by increasing salt concentration in the solvent (salting in).
  • the solubility may increase as the salt concentration in the solvent decreases (salting out).
  • a simple composition e.g., a composition containing only one peptide and a solvent
  • the relationship between peptide solubility and a change of composition may be easy to determine.
  • a complex composition such as a pharmaceutical or vaccine composition
  • the impact of a change in composition on peptide solubility may be difficult to determine.
  • a change in a pharmaceutical composition may simultaneously increase peptide solubility through one mechanism and decrease peptide solubility through another mechanism; for example, increasing the salt content in a composition may decrease peptide solubility by increasing ionic strength (salting out), but at the same time, may increase peptide solubility due to a change in pH.
  • One aspect of the present disclosure relates to a method for increasing peptide solubility.
  • the present disclosure related to a method for increasing the solubility of a neo-antigenic peptide. It was unexpectedly discovered that reducing the concentration of a pH modifier in a composition provided herein may increase the solubility of at least one neo- antigenic peptide.
  • the method of increasing peptide solubility comprises adjusting or reducing the concentration of a pH modifier to less than 1.0 mM.
  • the method of increasing peptide solubility comprises adding a pH modifier to a composition that comprises at least one neo-antigenic peptide and a pharmaceutically acceptable carrier such that the concentration of the pH modifier is lower than 1.0, 0.95, 0.90, 0.85, 0.80, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0.35, 0.30, 0.25, 0.20, 0.15, 0.10 mM or less.
  • the solubility of at least one neo-antigenic peptide in a composition provided herein, at a defined temperature is increased by at least 0.5, 1, 2, 3, 4, 5, 6,
  • the solubility of at least one neo-antigenic peptide in a composition provided herein, at a defined temperature is increased about 10% to about 300%, about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 200% to about 300%, about 250% to about 300%.
  • the solubility of at least one neo-antigenic peptide in a composition provided herein, at a defined temperature is increased about 1.1 to about lO-fold, about 1.5 to about lO-fold, about 2 to about lO-fold, about 3 to about lO-fold, about 4 to about lO-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about
  • the defined temperature is ambient temperature.
  • the defined temperature is at about 0°C, about l°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about l0°C, about l5°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C.
  • At least one neo-antigenic peptide in a pharmaceutical composition provided herein is insoluble, partially soluble or soluble in the composition at a given temperature.
  • At least one neo-antigenic peptide in a composition provided herein is soluble in the composition at ambient temperatures. In some embodiments, at least one neo-antigenic peptide in a composition provided herein is soluble in the composition at about 0°C, about l°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about l0°C, about l5°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, or about l00°C.
  • At least one neo- antigenic peptide in a composition provided herein is soluble in the composition at about 35°C, about 35.5°C, about 36°C, about 36.5°C, about 36.7°C, about 36.8°C, about 36.9°C, about 37°C, about 37. l°C, about 37.2°C, about 37.3°C, about 37.4°C, about 37.5°C, about 37.6°C, about 37.7°C, about 37.8°C, about 37.9°C, about 38°C, about 38.5°C, about 39°C, about 39.5°C, or about 40°C.
  • a pharmaceutical composition comprising at least one neo-antigenic peptide.
  • the present disclosure also directed to a vaccine composition and a peptide solution that comprise at least one neo-antigenic peptide.
  • a pharmaceutical composition of the present disclosure may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • “peptide” or“neo-antigenic peptide” as used herein includes the pharmaceutically acceptable salts thereof and the lyophilized form of the peptide.
  • Neo- antigenic peptides may be identified and synthesized by any suitable method known in the art or described herein. In some embodiments, the at least one neo-antigenic peptide contains about 5,
  • the at least one neo-antigenic peptide ranges from about 5 to about 50, from about 5 to about 45, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 25, from about 10 to about 50, from about 10 to about 45, from about 10 to about 40, from about 10 to about 35, from about 10 to about 30, from about 10 to about 25, from about 15 to about 50, from about 15 to about 45, from about 15 to about 40, from about 15 to about 35, from about 15 to about 30, from about 15 to about 25, from about 20 to about 50, from about 20 to about 45, from about 20 to about 40, from about 20 to about 35, from about 20 to about 30, or from about 20 to about 25 amino acids in length.
  • the at least one neo-antigenic peptide ranges from about 6 to about 25, from about 15 to about 35, from about 15 to about 24, from about 9 to about 15, from about 8 to about 11, or from about 9 to about 10 amino acids in length. In some embodiments, the length of the at least one neo-antigenic peptide is equal or less than 30, 25, 20, or 15 amino acid residues. In some embodiments, the length of each neo- antigenic peptide is independent of the length of other neo-antigenic peptides, if any, comprising the pharmaceutical composition.
  • each peptide in the present composition may be determined by one skilled in the art based on the particular application of the pharmaceutical composition.
  • the concentration and amount of each peptide may vary due to the condition of the subject, the administration method, and/or the solubility of the peptide.
  • each of the one to five neo-antigenic peptides or pharmaceutically acceptable salts thereof is present at a concentration of about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 pg/ml, 1000 pg/ml, 2000 pg/ml, 3000 pg/ml, 4000 pg/ml or above.
  • the total concentration of neo-antigenic peptides in the pharmaceutical composition is about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500 pg/ml or above.
  • each of the one or more neo-antigenic peptides or pharmaceutically acceptable salts thereof are present in the pharmaceutical composition at a concentration of from 10 pg/mL to 4000 pg/mL, 10 pg/mL to 3000 pg/mL, 10 pg/mL to 2000 pg/mL, 10 pg/mL to 1000 pg/mL, 10 pg/mL to 500 pg/mL, 10 pg/mL to 300 pg/mL, 25 pg/mL to 500 pg/mL, or 50 pg/mL to 300 pg/mL.
  • the at least one neo-antigenic peptide may or may not be soluble in the pharmaceutical composition.
  • the solubility of each neo-antigenic peptide is independent of the solubility of other neo-antigenic peptides.
  • the solubility of each neo-antigenic peptide is dependent of the solubility of other neo-antigenic peptides.
  • each of the neo-antigenic peptides may be soluble in the pharmaceutical composition.
  • the present disclosure relates to improving the solubility of at least one peptide; thus, in some embodiments, a higher percentage of at least one neo-antigenic peptide are soluble in a pharmaceutical composition than a percentage of the at least one neo- antigenic peptide in a pharmaceutical composition comprising the same pH modifier present at a concentration of 1.0 mM or higher and the same pharmaceutically acceptable carrier.
  • the percentage of the at least one neo-antigenic peptide soluble in a pharmaceutical composition is about 5% to about 100%, about 10% to about 100%, about 15% to about 100%, about 20% to about 100%, about 25% to about 100%, about 30% to about 100%, about 35% to about 100%, about 40% to about 100%, about 45% to about 100%, about 50% to about 100%, about 55% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, 95% to about 100%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%
  • all the neo-antigenic peptides are not soluble. In some embodiments, at least one of the neo-antigenic peptides may be partially soluble in the pharmaceutical composition. In some embodiments, at least one of the neo-antigenic peptides may be insoluble in the pharmaceutical composition. In some embodiments, at least one of the neo-antigenic peptides may remain insoluble in the pharmaceutical composition in order to achieve controlled release or other application needs. In some embodiments, at least one of the neo-antigenic peptides may be stabilized in an emulsion or suspension.
  • a pharmaceutical composition that comprises at least one soluble peptide and a method of selecting the soluble peptide are provided herein.
  • the solubility of a peptide may be predicted based on its HYDRO and pi values.
  • the at least one neo-antigenic peptide is bounded by pl>0 and HYDRO ⁇ -8, pi >0 and HYDRO >-4, or pl>4.3 and -4 ⁇ HYDRO>-8.
  • the at least one of the one or more neo-antigenic peptides is bounded by pi ⁇ 4.3 and -4 ⁇ HYDRO>-8.
  • the at least one neo-antigenic peptide is bounded by pi >0 and HYDRO >-4, or pl>4.3 and HYDRO ⁇ -4. In some embodiments, the at least one neo-antigenic is bounded by pi >0 and HYDRO >-4, or pl>4.3 and -4 ⁇ HYDRO>-9. In some embodiments, the at least one neo-antigenic peptide is bounded by 5 ⁇ pl>l2 and -4 ⁇ HYDRO>-9. In some embodiments, the at least one neo-antigenic peptide is bounded by pi >5 and HYDRO >-6.
  • the at least one neo-antigenic peptide is bounded by pl>8 and HYDRO >-8. In some embodiments, the at least one neo-antigenic peptide is bounded by pl ⁇ 5 and HYDRO >-5. In some embodiments, the at least one neo-antigenic peptide is bounded by pl>9 and HYDRO ⁇ -8 In some embodiments, the at least one neo-antigenic peptide is bounded by pi >7 and a HYDRO value of >-5.5.
  • a method of selecting soluble peptides for the pharmaceutical composition may comprise determining the pi and HYDRO values of a peptide and comparing its pi and HYDRO values with the pi and HYDRO ranges provided herein.
  • Pharmaceutically acceptable salts may refer to appropriate salts or complexes of active compounds which retain the desired biological activity of the parent compound and exhibit limited toxicological effects to normal cells.
  • a non-exhaustive list of example of such salts include (a) acid addition salts formed with inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like), and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and polyglutamic acid, etc; and (b) base addition salts formed with metal cations such as zinc, calcium, sodium, potassium, and the like, among numerous others.
  • Each one of the neo-antigenic peptides may be independently modified to affect its efficacy, solubility, stability, bioavailability, the rate of metabolism, and/or other properties. Modifications may be carried out at either a side-chain or a terminus by chemical means or by conjugating the peptide or peptides with one or more carriers (e.g., proteins).
  • Each type of the neo-antigenic peptide may contain one or more chiral centers and thus occur as stereoisomers, racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomers mixtures, and/or cis-trans isomers. All such isomeric forms of these compounds are expressly included herein.
  • the compounds of this disclosure may be also present in multiple tautomeric, amorphous and/or crystal forms, and all the tautomeric, amorphous and crystal forms of the compounds are expressly included in the present disclosure.
  • a pH modifier refers to a compound or a combination of two or more compounds that, when added to a pharmaceutical composition, is capable of changing the pH of the composition.
  • a pH modifier may comprise an acid, organic acid, inorganic acid, latent acid, weak acid, base, conjugate base, organic base, inorganic base, latent base, a pharmaceutically acceptable salt of an acid or base, and/or a combination thereof.
  • a latent acid or base is a compound that may become an acid or base at particular conditions such as after heating or in the presence of water.
  • a pH modifier may be in solid and/or liquid form and may be soluble, partially soluble, or insoluble in the pharmaceutical composition.
  • the pH modifier comprises an organic acid and/or a pharmaceutically acceptable salt thereof.
  • a suitable organic acid may contain one or more acidic groups such as carboxylic, sulfonic, phosphoric, phosphorous, and hypophosphorous acid groups.
  • suitable acids that may be comprised in a pH modifier include mono-, di-, tri- or polybasic carboxylic acid, mono-, di-, tri- or polybasic sulfonic acid, Sorbic acid, adipic acid, malonic acid, succinic acid, glutaric acid, maleic acid, fumaric acid, gluconic acid, lactic acid, glycolic acid, ascorbic acid, malic acid, tartaric acid, tartronic acid, mucic acid, citric acid, amino acid, glutamic acid, aspartic acid, acids containing aromatic groups such as benzoic acid, phthalic acid, isophthalic acid, salicylic acid, terephthalic acid, and trimellitic acid, and polymeric acid such as poly(acrylic acid) and poly(methacrylic acid).
  • the pH modifier comprises a base and/or a pharmaceutically acceptable salt thereof, such as compounds containing amino groups, hydroxyl groups, amidine groups, or phosphazene groups.
  • the pH modifier comprises a dicarboxylate or tri carboxyl ate salt.
  • the pH modifier is succinic acid, monosodium succinate, disodium succinate or a combination thereof.
  • the disodium succinate is disodium succinate hexahydrate.
  • the pH modifier is citric acid, disodium citrate, trisodium citrate or a combination thereof. Cations of salts comprising a pH modifier may independently be chosen for each salt, and the cations can be organic or inorganic.
  • Non-exhaustive examples of cations include sodium, aluminum, calcium, lithium, magnesium, zinc, potassium, arginine, benzathine, chloroprocaine, choline, diethanolamine, ethanolamine, ethylenediamine, histidine, lysine, procaine, and trimethylamine.
  • the concentration of the pH modifier may be less than 1.0 mM. It has been unexpectedly discovered that a pH modifier concentration lower than 1.0 mM provides improved neo- antigenic peptide solubility in the pharmaceutical composition provided herein compared to a pH modifier concentration equal or higher than 1.0 mM.
  • the pH modifier is present in the pharmaceutical composition at a concentration of lower than or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19,
  • the pH modifier is present in the pharmaceutical composition at a concentration of lower than 1.0 mM and higher than about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, or 0.95 mM.
  • the pH modifier is present in the pharmaceutical composition at a concentration of lower than about 0.75 mM and higher than about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, or 0.70 mM. In some embodiments, the pH modifier is present in the pharmaceutical composition at a concentration of lower than about 0.50 mM and higher than about 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, or 0.45 mM. It is to be understood that depending on the properties of a pH modifier (e.g., pKa value), the appropriate concentration for one pH modifier may not be appropriate for a different pH modifier. When one skilled in the art substitutes a pH modifier in the present composition, the optimum concentration of the pH modifier required may change accordingly.
  • Acceptable carriers as provided herein are physiologically acceptable to be administered to patients, and should retain the therapeutic properties of the active ingredient, compound or drug with/in which it is administered. Acceptable carriers and their formulations are generally described in, for example, Remington’ Pharmaceutical Sciences (18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990).
  • a pharmaceutically acceptable carrier is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the active ingredients from the administration site of one organ, or portion of the body, to another organ, or portion of the body, or in an in vitro assay system.
  • Acceptable carriers are compatible with other ingredients of the pharmaceutical composition and do not substantially alter the specific activity of the neo-antigenic peptides. At the dosages and concentrations employed, acceptable carriers should be non-toxic and not injurious to a subject to whom it is administered. Depending on the application and route of administration, an acceptable carrier may be in a gas, liquid, or solid form.
  • An acceptable carrier may refer to a solvent, a co-solvent, a buffer, an isotonic agent, a conjugating or complexing agent, ionic and/or non-ionic surfactants, viscosity modifiers, systems that contain emulsions, suspensions, liposomes, polymeric micelles, microspheres, porous nanostructures, dendrimers, lipid-based nanoparticles, gels, cyclodextrins, carrier proteins, etc, or a combination thereof.
  • a conjugating or complexing agent may be a compound that interacts, either chemically or physically, with a component of the composition such that the stability, solubility, or other properties of that component may be altered; for example, a compound that interacts and increases the solubility of at least one neo-antigenic peptide.
  • Conjugating or complexing agents include but are not limited to chelating agents like EDTA, proteins such as keyhole limpet hemocyanin, bovine serum albumin, ovalbumin, gelatin, and immunoglobulins, cyclic oligosaccharides like cyclodextrins, and metal complexes like Zn- protein complexes.
  • a system that contains emulsions, suspensions, liposomes, polymeric micelles, microspheres, porous nanostructures, dendrimers, lipid-based nanoparticles, gels, cyclodextrins, carrier proteins, etc, or a combination thereof refers to a composition that comprises any one of the abovementioned components or mechanisms.
  • the pharmaceutical composition comprises a buffer.
  • a buffer refers to a solution, the pH of which is negligibly altered when a small amount of acidic or basic material is added into it.
  • Exemplary buffering agents include but are not limited to phosphate, citrate, citric acid, acetic acid, borate, and histidine.
  • the present composition comprises a surfactant.
  • the surfactant may be non-ionic, anionic, cationic, amphoteric, zwitterionic, or a combination thereof.
  • Exemplary surfactants include but are not limited to ethers of fatty alcohols and/or polyoxyalkylene alkyl ethers, polyoxyalkylene fatty acid esters, Tweens®, polysorbates, Pluronics®, and poloxamers, polyoxyalkylenes.
  • the pharmaceutically acceptable carrier comprises water and/or at least one non-aqueous liquid.
  • acceptable non-aqueous liquids include but not limited to DMSO, DMF, acetonitrile, alcohols (e.g., ethanol) and a combination thereof.
  • the pharmaceutically acceptable carrier comprises a sugar, which includes but is not limited to, monosaccharides such as dextrose, glucose, fructose, and galactose, and disaccharides such as sucrose, lactulose, lactose, maltose, trehalose, cellobiose, and chitobiose.
  • the pharmaceutically acceptable carrier may also comprise a polysaccharide such as starch, cellulose, and chitin, and sugar alcohols such as mannitol and sorbitol.
  • the pharmaceutically acceptable carrier comprises water and dextrose.
  • the amount and concentration of a pharmaceutically acceptable carrier in the pharmaceutical composition may be dependent on the properties of other components herein; for example, a higher concentration of DMSO may be suitable for a pharmaceutical composition comprising more hydrophobic neo- antigenic peptides.
  • the concentration of DMSO present in the pharmaceutical composition is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or above by volume (% v/v).
  • the concentration of the sugar present in the pharmaceutical composition is about 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% or above in water by weight (% w/w water). In some embodiments, the concentration of the sugar present in the composition is about 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% or above by weight of the solution (% w/w).
  • the concentration of the sugar present in the composition is about 1%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5% or above by weight of water in the solution (% w/w water).
  • the present pharmaceutical composition may further comprise other additives, such as diluents, fillers, binders, disintegrants, lubricants, coloring agents, preservatives, stabilizers, and agents facilitating delayed release, the presence and amount of which may be dependent on the route of administration.
  • Compositions provided herein may be administered orally, topically, parenterally such as by intravenous, intramuscular, subcutaneous, intra-arterial, intra-articular, intrathecal, and intradermal administration, or by other applicable means of administration.
  • the present composition may be administered by injection of a unit dose.
  • the present pharmaceutical composition may be a vaccine composition, wherein at least one neo-antigenic peptide can be utilized as a vaccine.
  • a vaccine composition may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or more neo- antigenic peptides.
  • the composition comprises an immunomodulator or adjuvant.
  • the present composition comprises two or more immunomodulators or adjuvants.
  • Immunomodulator may refer to an agent that induces, amplifies, attenuates, prevents, or otherwise changes the immune response or the functioning of the immune system.
  • An adjuvant may refer to an agent that modifies the immunological function of another agent.
  • the immunomodulator or adjuvant provided herein may comprise poly-ICLC, polyinosinic:polycytidylic acid (poly I:C, or poly I: poly C), 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, ARNAX, STING agonists, dSLIM, GM- CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryllipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector system, PLGA microparticles,
  • the adjuvant may also comprise an acrylic (co)polymer, a methacrylic (co)polymer, and/or a copolymer of maleic anhydride and an alkenyl derivative.
  • a (co)polymer may be a polymer of acrylic or methacrylic acid cross-linked with a polyalkenyl ether of a sugar or polyalcohol (carbomer), a polymer of acrylic or methacrylic acid cross-linked with an allyl sucrose or with allylpentaerythritol, a copolymer of maleic anhydride and ethylene cross-linked with divinyl ether.
  • an immunomodulator or adjuvant may comprise cytokines (such as TNF- alpha, GM-CSF, IL-l, IL-4, and IL-12), toll like receptors (such as TLR4 and TLR9), chemically modified CpGs (e.g., CpR, Idera), non-CpU bacterial DNA or RNA, and immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX- 4016, sildenafil, tadalafil, vardenafil, sorafmib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175.
  • cytokines such as TNF- alpha, GM-CSF, IL-l, IL-4, and IL-12
  • toll like receptors such
  • the immunomodulator or adjuvant comprises poly-ICLC (poly-ICLC).
  • the immunomodulatory(s) or adjuvant(s) may be administered separately to the vaccine or pharmaceutical composition; alternatively, they may be administered separately but in parallel with the present composition.
  • the pharmaceutical composition comprises: (a) one to five neo- antigenic peptides or pharmaceutically acceptable salts thereof; (b) DMSO; (c) sugar such as dextrose; (d) lower than 1.0 mM succinic acid or a succinate salt; (e) poly I: poly C; (f) poly-L- lysine; (g) carboxymethylcellulose, (h) chloride; and (i) water.
  • each of the one to five peptides or pharmaceutically acceptable salts thereof is present at a concentration of about 50, 100, 150, 200, 250, 300, 350, 400 pg/ml or above.
  • the pharmaceutical composition comprises equal or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% DMSO by volume (% v/v). In some embodiments, the DMSO is present in the pharmaceutical composition between about 1% to 3% by volume (% v/v). In some embodiments, the pharmaceutical composition comprises dextrose at a concentration of about 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%,
  • the pharmaceutical composition comprises dextrose at a concentration of about 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%,
  • the pharmaceutical composition comprises about 3.6-3.7% dextrose by weight of the solution (% w/w). In some embodiments, the pharmaceutical composition comprises about 3.6-3.7% dextrose by weight of the water in the solution (%w/w water). In some embodiments, the pharmaceutical composition comprises poly Lpoly C at a concentration of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg/ml or above.
  • the pharmaceutical composition comprises poly-L-lysine at a concentration of about 0.100, 0.125, 0.150, 0.175, 0.200, 0.225, 0.250, 0.275, 0.300, 0.325, 0.350, 0.375, 0.400, 0.425, 0.450, 0.475, 0.500 mg/ml or above.
  • the pharmaceutical composition comprises sodium carboxymethylcellulose at a concentration of about 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, 2.50 mg/ml or above.
  • the pharmaceutical composition comprises sodium chloride at a concentration of 0.100%, 0.125%, 0.150%, 0.175%, 0.200%, 0.225%, 0.250%, 0.275%, 0.300%, 0.325%, 0.350%, 0.375%, 0.400%, 0.425%, 0.450%, 0.475%, 0.500% or above by weight of the water in the solution (%w/w water).
  • the pharmaceutical composition comprises sodium chloride at a concentration of 0.100%, 0.125%, 0.150%, 0.175%, 0.200%, 0.225%, 0.250%, 0.275%, 0.300%, 0.325%, 0.350%, 0.375%, 0.400%, 0.425%, 0.450%, 0.475%, 0.500% or above by weight of the solution (% w/w).
  • the pharmaceutical composition may be lyophilizable or lyophilized.
  • lyophilized compositions may have longer shelf life and may be more convenient to transport.
  • the shelf life of the lyophilized compositions is about 0.5 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes or about 60 minutes.
  • the shelf life of the lyophilized compositions is about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, or about 24 hours.
  • the shelf life of the lyophilized compositions is about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 28 days, about 30 days, or about 31 days. In some embodiments, the shelf life of the lyophilized compositions is about 1 month, about 2 months, about 3 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months. In some embodiments, the shelf life of the lyophilized compositions is about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years.
  • the shelf life of the lyophilized compositions is in the range of about 0.5 minutes to about 60 minutes, 1 minute to about 60 minutes, 2 minutes to about 60 minutes, 3 minutes to about 60 minutes, 4 minutes to about 60 minutes, 5 minutes to about 60 minutes, 10 minutes to about 60 minutes, 15 minutes to about 60 minutes, 20 minutes to about 60 minutes, 30 minutes to about 60 minutes, or 45 minutes to about 60 minutes.
  • the shelf life of the lyophilized compositions is in the range of about 1 hour to about 24 hours, 2 hours to about 24 hours, 3 hours to about 24 hours, 4 hours to about 24 hours, 5 hours to about 24 hours, 10 hours to about 24 hours, 15 hours to about 24 hours, or 20 hours to about 24 hours.
  • the shelf life of the lyophilized compositions is in the range of about 1 day to about 31 days, 2 days to about 31 days, 3 days to about 31 days, 4 days to about 31 days, 5 days to about 31 days, 10 days to about 31 days, 15 days to about 31 days, 20 days to about 31 days, 25 days to about 31 days. In some embodiments, the shelf life of the lyophilized compositions is in the range of about 1 month to about 12 months, 2 months to about 12 months, 3 months to about 12 months, 4 months to about 12 months, 5 months to about 12 months, 6 months to about 12 months, 7 months to about 12 months, 8 months to about 12 months, 9 months to about 12 months, 10 months to about 12 months, or about 11 months to about 12 months.
  • the shelf life of the lyophilized compositions is in the range of about 1 year to about 2 years, about 1 year to about 3 years, about 1 year to about 4 years, about 1 year to about 5 years, about 2 years to about 3 years, about 2 years to about 4 years, about 2 years to about 5 years, about 3 years to about 4 years, about 3 years to about 5 years, or about 4 years to about 5 years.
  • the shelf life of the lyophilized pharmaceutical composition may be increased about 10% to about 300%, about 20% to about 300%, about 50% to about 300%, about 100% to about 300%, about 150% to about 300%, about 200% to about 300%, about 250% to about 300%.
  • the shelf life of the lyophilized pharmaceutical composition may be increased about 1.1 to about lO-fold, about 1.5 to about lO-fold, about 2 to about lO-fold, about 3 to about lO-fold, about 4 to about lO-fold, about 1.1 to about 5-fold, about 1.1 to about 6-fold, about 1.1 to about 7-fold, about 1.1 to about 8-fold, about 1.1 to about 9-fold, about 2 to about 5-fold, about 2 to about 6-fold, about 2 to about 7-fold, about 2 to about 8-fold, about 2 to about 9-fold, about 3 to about 6-fold, about 3 to about 7-fold, about 3 to about 8-fold, about 3 to about 9-fold, about 4 to about 7-fold, about 4 to about 8-fold, about 4 to about 9-fold, at least about 1.1 -fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold,
  • one or more components of the present composition may be lyophilized.
  • the present composition comprises at least one lyophilized peptide.
  • the present composition comprises at least one lyophilized peptide and a lyophilized pH modifier.
  • One or more lyophilized components may be combined with a sterile solution prior to administration.
  • at least one lyophilized peptide may be combined with a non-lyophilized pharmaceutically acceptable carrier before administration.
  • at least one lyophilized peptide may be combined with a non-lyophilized pH modifier and a non-lyophilized pharmaceutically acceptable carrier before administration.
  • at least one lyophilized peptide may be combined with a non-lyophilized pH modifier, a non-lyophilized immunomodulatory or adjuvant and a non-lyophilized pharmaceutically acceptable carrier before administration.
  • a method of preparing a pharmaceutical composition is also provided herein.
  • a method of preparing the present composition may comprise combining at least one peptide with a pH modifier and a pharmaceutically acceptable carrier.
  • a method of preparing the present composition may comprise combining at least one peptide with a pH modifier, an immunomodulatory or adjuvant and a pharmaceutically acceptable carrier.
  • a method of preparing the present composition may also comprise combining a peptide with a pH modifier and a pharmaceutically acceptable carrier such that the concentration of the pH modifier is less than 1.0 mM.
  • a method of preparing the present composition may also comprise combining a peptide with a pH modifier, an immunomodulatory or adjuvant and a pharmaceutically acceptable carrier such that the concentration of the pH modifier is less than 1.0 mM.
  • a method of preparing a pharmaceutical composition may comprise the preparation of a solution that comprises at least one neo-antigenic peptide. The preparation of such a solution may refer to the dissolution of the at least one peptide into a solvent; for example, dissolving a peptide into DMSO in order to make a 50 mg/ml peptide in DMSO stock.
  • Steps or procedures that are commonly used in handling a solution may include agitating, mixing, shaking, sonicating, heating, cooling, diluting, filtering, etc, and one having ordinary skill in the art may recognize the suitable steps or procedures and when additional step or procedure will be necessary for a particular peptide or solution.
  • at least one peptide is first dissolved in DMSO, and the DMSO stock is combined with an aqueous solution that comprises the pH modifier.
  • at least one peptide and a pH modifier are dissolved into a solution simultaneously.
  • one or more filtration steps may be employed to remove any insoluble materials before and/or after any combination of components of the present composition.
  • a method of preparing a neo-antigenic peptide solution is provided herein.
  • the preparation of such a solution may refer to the dissolution of the at least one peptide into a solvent; for example, dissolving a peptide into DMSO in order to make a stock of 50 mg/ml peptide in DMSO.
  • a method of preparing the present solution may also comprise combining a peptide with a pH modifier such that the concentration of the pH modifier is less than 1.0 mM.
  • At least one peptide is first dissolved in DMSO, and the DMSO stock is combined with an aqueous solution that comprises the pH modifier. In other embodiments, at least one peptide and a pH modifier are dissolved into a solution simultaneously. In some embodiments, one or more filtration steps may be employed to remove any insoluble materials before and/or after any combination of components of the present composition.
  • kits comprising one or more of the components of the formulation is provided.
  • the kits may comprise instructions that provide users with information such as how to use the kits and how to administer the resulted formulation. If applicable, each component of the present composition may be included in the kits as a liquid, a solid, or a combination thereof; for example, the at least one peptide and/or the pH modifier may be pre-dissolved in a solution that is provided in the kit, or it may be in solid form that users may need to combine it with a liquid before administration, depending on the particular instruction provided with the kits.
  • kits comprise a lyophilized composition that comprises the at least one neo- antigenic peptide.
  • the kits may comprise instructions that teach users to combine the at least one peptide with a pH modifier such that the pH modifier is present at a concentration of less than 1.0 mM in the resultant solution.
  • the kits may further comprise a pharmaceutically acceptable carrier such as water, DMSO, other (co-)solvent, or a combination thereof.
  • kits may comprise various containers (such as a vial, a bottle, a tube, a bag, and a sheet of (non-)dissolvable paper), various liquids (such as solvent, co-solvent, and buffer), and various solids (such as salts, sugars, and lyophilized peptides).
  • various containers such as a vial, a bottle, a tube, a bag, and a sheet of (non-)dissolvable paper
  • various liquids such as solvent, co-solvent, and buffer
  • various solids such as salts, sugars, and lyophilized peptides.
  • the kits contain a single dose or multiple doses that require no reconstitution, where a user may administer the formulation contained in the kit as is.
  • kits contain a single dose or multiple doses that require reconstitution, where a user may combine a component provided in the kits with other component(s), which may or may not be provided in the kits; for example, a user may need to combine a lyophilized peptide provided in the kits with water or buffer that may or may not be provided in the kits.
  • kits provided herein may comprise a viral vector.
  • the at least one neo-antigenic peptide may be encoded and expressed in cells either in vivo or in vitro.
  • viral vectors that may be used include vectors based on lentiviruses, vectors based on adenoviruses, vectors based on adeno associated viruses (AAV), vectors based on poxviruses such as vaccinia virus, ALVAC, Modified Vaccinia Ankara (MV A) virus, and modified Copenhagen strain of vaccinia virus, and vectors based on retroviruses such as murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), and human immuno deficiency vims (HIV).
  • MuLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno defic
  • a method of preparing a vaccine composition may comprise the preparation of a solution that comprises at least one neo-antigenic peptide.
  • the preparation of such a solution may refer to the dissolution of the at least one peptide into a solvent; for example, dissolving a peptide into DMSO in order to make a 50 mg/ml peptide in DMSO stock.
  • a method of preparing the vaccine composition may comprise combining a peptide with a pH modifier and a pharmaceutically acceptable carrier such that the concentration of the pH modifier is less than 1.0 mM.
  • a method of preparing the vaccine composition may also comprise combining the at least one peptide with an immunomodulator or adjuvant.
  • At least one peptide is first dissolved in DMSO; then the DMSO stock is combined with an aqueous solution that comprises the pH modifier; and lastly, a solution that comprises an immunomodulator or adjuvant is combined into the above peptide-pH modifier solution.
  • at least one peptide, a pH modifier, and an immunomodulator or adjuvant are dissolved into a solution simultaneously.
  • one or more filtration steps may be employed to remove any insoluble materials before and/or after any combination of components of the present composition.
  • a method of treating a condition or disease by administering a composition provided herein is also contemplated.
  • the condition or disease is neoplasia.
  • the neoplasia is a cancer.
  • the method of treating a subject with neoplasia may refer to inducing an immune response in a subject and treating the neoplasia in the subject by administering the subject a vaccine or pharmaceutical composition.
  • the compositions may be used for a subject that has been diagnosed as having neoplasia, or at risk of developing it.
  • the compositions may be administered to a subject in an amount and at an interval (in the case of a series of administrations) that is suitable for that subject.
  • the method of treating a condition or disease may comprise administering one unit dose of a composition to a subject at regular intervals such as a day, a week, and a month for a period of time.
  • the method comprises administering a 2 nd , 3 rd , 4 th , 5 th , 6 th , 7 th , 8 th , 9 th , l0 th , or more compositions to the subject, where each administration, including the composition, the amount, and timing of the administration, is independently chosen.
  • cancers that may be prevented or treated in accordance with the compositions include, but are not limited to: renal cancer, kidney cancer, glioblastoma multiforme, metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myclodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Ho
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas.
  • a composition may be administered to a subject with neoplasia immediately after preparing the composition.
  • a composition may be administered to a subject with neoplasia about 0.5 minutes, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes or about 60 minutes after preparing the composition.
  • a composition may be administered to a subject with neoplasia about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 10 hours, about 15 hours, about 20 hours, or about 24 hours after preparing the composition.
  • a composition may be administered to a subject with neoplasia about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 10 days, about 15 days, about 20 days, about 25 days, about 28 days, about 30 days, or about 31 days after preparing the composition.
  • a composition may be administered to a subject with neoplasia about 1 month, about 2 months, about 3 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after preparing the composition.
  • a composition may be administered to a subject with neoplasia about 1 year, about 2 years, about 3 years, about 4 years, or about 5 years after preparing the composition.
  • a composition may be administered to a subject with neoplasia in the range of about 0.5 minutes to about 60 minutes, 1 minute to about 60 minutes, 2 minutes to about 60 minutes, 3 minutes to about 60 minutes, 4 minutes to about 60 minutes, 5 minutes to about 60 minutes, 10 minutes to about 60 minutes, 15 minutes to about 60 minutes, 20 minutes to about 60 minutes, 30 minutes to about 60 minutes, or 45 minutes to about 60 minutes after preparing the composition.
  • a composition may be administered to a subject with neoplasia in the range of about 1 hour to about 24 hours, 2 hours to about 24 hours, 3 hours to about 24 hours, 4 hours to about 24 hours, 5 hours to about 24 hours, 10 hours to about 24 hours, 15 hours to about 24 hours, or 20 hours to about 24 hours after preparing the composition.
  • a composition may be administered to a subject with neoplasia in the range of about 1 day to about 31 days, 2 days to about 31 days, 3 days to about 31 days, 4 days to about 31 days, 5 days to about 31 days, 10 days to about 31 days, 15 days to about 31 days, 20 days to about 31 days, 25 days to about 31 days after preparing the composition.
  • a composition may be administered to a subject with neoplasia in the range of about 1 month to about 12 months, 2 months to about 12 months, 3 months to about 12 months, 4 months to about 12 months, 5 months to about 12 months, 6 months to about 12 months, 7 months to about 12 months, 8 months to about 12 months, 9 months to about 12 months, 10 months to about 12 months, or about 11 months to about 12 months after preparing the composition.
  • a composition may be administered to a subject with neoplasia in the range of about 1 year to about 2 years, about 1 year to about 3 years, about 1 year to about 4 years, about 1 year to about 5 years, about 2 years to about 3 years, about 2 years to about 4 years, about 2 years to about 5 years, about 3 years to about 4 years, about 3 years to about 5 years, or about 4 years to about 5 years after preparing the composition.
  • the present disclosure also contemplates a pharmaceutical or vaccine formulation for use in the preparation of a medicament for the treatment of a condition or disease.
  • the medicament may comprise the one or more lyophilized or nondyophilized components of the present composition, or a combination or a fraction thereof.
  • the present disclosure provides a process for the preparation of a medicament that comprises the present composition.
  • the condition or disease is neoplasia.
  • Peptides were chemically synthesized and purified. All peptides were weighed in 5 mL Eppendorf tubes using a XP105 Delta Range balance by Mettler Toledo. The theoretical peptide content of the lyophilized peptides was determined by weight, theoretical trifluoroacetic acid (TFA) content, and percent purity. The percent peptide content was determined using Equations 1 and 2; alternatively, it may be determined experimentally. The target gross weight (mg) was then calculated using Equation 3, and the volume of DMSO required to obtain a peptide concentration of 50 mg/ml was determined using Equation 4.
  • TFA trifluoroacetic acid
  • the succinate buffers used in this study were prepared daily by diluting 1M sodium succinate in 5% dextrose in water (D5W) solution to the desired concentration. After the peptides had been dissolved in DMSO to obtain a 50 mg/ml stock solution, 60 pL of the stock solution was then diluted with 1.44 mL of 5 mM, 0.75 mM, 0.5 mM, or 0.25 mM sodium succinate in D5W. The pH of each peptide solution was measured using a Gene Mate meter with electrode pHE-l l. The peptide solutions were then stored under ambient conditions for a minimum of 2 hours to allow any potential precipitation or gelation to occur. Peptides were considered completely soluble if the solutions appeared transparent and did not form a gel at the duration of the study.
  • Table 5 Formulation solubility results of 20 peptides insoluble in 5mM succinate, and which became soluble in 0.75 mM, 0.50 mM and 0.25 mM succinate.
  • Whether a peptide or peptide mixture is completely soluble in a formulation at a given temperature can be determined using turbidity measurements.
  • Pharmaceutical formulations containing a peptide or peptide mixture shall be prepared using the methods outlined in Example 1.
  • reference formulations approximately identical in composition to the pharmaceutical formulations except for the absence or the peptide or peptide mixture shall be prepared.
  • the turbidity of the reference and pharmaceutical formulations shall then be measured using any instrument or technique suitable for measuring turbidity.
  • Such instruments and techniques include, but are not limited to, visual inspection of the formulations, a turbidimeter or a turbidity sensor.
  • Example 3 demonstrates that the present composition is compatible with poly-ICLC (poly ICLC), an RNA-based immunostimulant. While the lower succinate concentration is a useful universal formulation for peptide solubility, there were hypotheses that it may not be compatible with poly-ICLC because peptides in lower succinate concentrations have a pH of 3.2- 4.0 and poly-ICLC may precipitate when combined with the lower pH peptides solution.
  • the approximate pKa of the phosphate groups in RNA is 2 and poly ICLC already exists as a cloudy suspension; thus, it is possible that the lower pH of the formulation may cause the poly ICLC to further precipitate or aggregate.
  • poly ICLC was combined with various peptide combinations in 5 mM, 0.50 mM, and 0.25 mM succinate to show that poly-ICLC is compatible with the present formulations comprising a pH modifier at less than 1.0 mM concentration.
  • Table 6 pH values of individual and combined peptides, which are soluble in 5 mM, 0.5 mM and 0.25 mM succinate
  • “Design 1” contains three peptide pools where pool 1 contains three peptides (i.e., L7, L8 and L14), pool 2 contains two peptides (i.e., L9 and LlOc) and pool 3 contains two peptides (i.e., L15 and Ll lf).
  • “Design 6” contains two peptide pools where pool 1 contains four peptides (i.e., L7, L8, L9 and L14) and pool 2 contains two peptides (i.e., L15 and Ll lf).
  • “Design 10” contains four peptide pools where pool 1 contains five peptides (i.e., L7, L8, L9, LlOc and L14), pool 2 contains one peptide (i.e., L15), pool 3 contains one peptide (i.e., Ll lf) and pool 4 contains one peptide (i.e., Ll li).
  • concentration of each peptide in the pools can be changed according to one skilled in the art of preparing peptide formulations.
  • Table 8 Description of GATA3 Pool Designs
  • C- terminal histidine was incorporated into the sequence by being preloaded onto the resin by using either H-His(Trt)-2Cl-Trt resin or Fmoc-His(Trt)-Wang (LL) resin.
  • Fmoc-Asp(OMpe)-OH may be used in place of Fmoc-Asp(OtBu)-OH to help improve synthesis, such as with sequence combinations of“DG” to minimize aspartamide formation.
  • the peptide sequences were swelled with dimethylformamide (DMF) and drained twice. Synthesis began with the deprotection of the N-a-FMOC protecting group using 20% piperidine in DMF with nitrogen dispensing to mix. After draining, the resin was washed with DMF. Next, 6 mL 0.4 M amino acid solution was added along with 5.8 mL 0.4 M HCTU and 6 mL of 0.8 M DIEA for 20 minutes. The coupling reaction was run with nitrogen dispensing to mix, followed by draining the reaction vessel (RV). The amino acid, HCTU, and DIEA additions were repeated for a double coupling cycle with the same mixing and draining parameters as the first coupling step.
  • DMF dimethylformamide
  • the resin was then washed with DMF again. This cycle was repeated for every amino acid residue.
  • the final deprotection method removed the N-terminal Fmoc via 20% piperidine in DMF, and the resin was washed with DMF followed by washes with MeOH. The resin was on the instrument under nitrogen until removed.
  • This coupling reaction followed 105 °C / 288W heating followed by 105 °C / 73 W heating.
  • This first deprotection was initially diluted with DMF, however this step was not required for any subsequent deprotections as the RV already contained DMF from the coupling reaction.
  • the deprotection, wash, and coupling cycles were repeated for each residue until the peptide had been synthesized.
  • a double coupling step was performed, where after the single coupling was performed, the solution was drained, and the coupling step was repeated before proceeding to the deprotection.
  • the final deprotection of the N-terminal Fmoc group was performed as above, except for being drained and washed twice with DMF before being transferred via DMF back to the original HT resin position.
  • the resin was transferred to a fritted syringe using DMF, rinsed with MeOH, and dried using a vacuum manifold. Then the resin was cleaved using Reagent K (82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol, and 2.5% ethanedithiol) using an upright holder on an oscillating shaker for three hours at room temperature.
  • Reagent K 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol, and 2.5% ethanedithiol
  • the cleavage cocktail was then dispensed through a filtered syringe frit into cold diethyl ether or cold methyl tert- butyl ether (MTBE). Each syringe was then rinsed with a 95:5 trifluoroacetic acid:water solution by agitation. The rinse was then added to the rest of the cocktail/ether mixture. The mixture was then centrifuged. After decanting the ether, another cold ether wash was added. The container was vortexed and centrifuged again. This was repeated to thoroughly rinse the pellet. The final wash was decanted and the pellet dried via vacuum desiccator.
  • MTBE cold methyl tert- butyl ether
  • a sample of the pellet was dissolved in solvent (e.g ., DMSO, DMF, water, or acetonitrile) and analyzed via ETPLC-MS for identity, crude purity, and retention time.
  • solvent e.g ., DMSO, DMF, water, or acetonitrile
  • Other peptides for example L14 (SMLTGPPARVPAVPFDLH), L8 (GPP ARVP AVPFDLHF CRS SIMKPKRD), LlOc (KPKRDGYMFLKAESKI), Ll lh (FLKAESKIMFATLQR), and Ll li (E SKIMF ATLQRS SL) were made in a similar fashion, using amino acids and pre-loaded resins specific to those sequences.
  • Fmoc-amino acids were used in synthesizing peptide L15 (KPKRDGYMFLK AESKIMF ATLQRS SLW CLC SNH) : Fmoc-Ala-OH-H 2 0, Fmoc-Cys(Trt)- OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-Asn(Trt)-OH, Fmoc-Pro-OH, Fmoc- Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(t
  • C-terminal histidine was incorporated into the sequence by being preloaded onto the resin by using either H-His(Trt)-2Cl-Trt resin or Fmoc-His(Trt)-Wang (LL) resin.
  • Fmoc-Asp(OMpe)-OH may be used in place of Fmoc-Asp(OtBu)-OH to help improve synthesis, such as with sequence combinations of “DG” to minimize aspartamide formation.
  • amino acid dipeptides (psuedoprolines) were incorporated to improve synthesis yields, such as Fmoc-Ser(tBu)- Ser(psi(Me,Me)pro)-OH in place of“SS”, Fmoc-Ala-Thr(psi(Me,Me)pro)-OH in place of“AT”, and Fmoc-Glu(OtBu)-Ser(psi(Me,Me)pro)-OH in place of“ES”.
  • the programmed Fmoc-amino acid was then added (0.5 M in DMF) to the RV along with 4 M DIC and 0.25 M Oxymapure. This coupling reaction followed 105 °C / 288W heating followed by 105 °C / 73 W heating. The deprotection, wash, and coupling cycles were repeated for each residue until the peptide had been synthesized. For arginine residues, there was a double coupling step, where after the single coupling was performed, the solution was drained, and the coupling step was repeated before proceeding to the deprotection. The final deprotection of the N-terminal Fmoc group was performed the same as all other deprotections steps, except for being drained and washed twice with DMF before being transferred via DMF back to the original HT resin position.
  • the resin was transferred to a fritted syringe using DMF, rinsed with MeOH, and dried using vacuum manifold. The resin was then cleaved using Reagent K (82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol, and 2.5% ethanedithiol) using an upright holder on an oscillating shaker at room temperature.
  • Reagent K 82.5% trifluoroacetic acid (TFA), 5% water, 5% thioanisole, 5% phenol, and 2.5% ethanedithiol
  • the cleavage cocktail was then dispensed through filtered syringe frit into cold diethyl ether (or cold MTBE). Each syringe was then rinsed with a 95:5 trifluoroacetic acid: water solution by agitation. The rinse was then added to the rest of the cocktail/ether mixture. Then the mixture was centrifuged. After decanting the ether, another cold ether wash was added. The container was vortexed and centrifuged again. This was repeated to thoroughly rinse the pellet. The final wash was decanted and the pellet was dried via vacuum desiccator. A sample of the pellet was dissolved in solvent (e.g., DMSO, DMF, water, or acetonitrile) and analyzed via UPLC-MS for identity, crude purity, and retention time.
  • solvent e.g., DMSO, DMF, water, or acetonitrile
  • a number of GATA3 peptides with neoepitopes were first tested for solubility using 5 mM sodium succinate (SS) in D5W with 4% DMSO. Based on the initial results the formulation strategy was improved by adjusting the sodium succinate (SS) concentration and DMSO amount, which lead to the selection of 7 peptides.
  • the pooling strategies of these peptides were determined for solubility and compatibility with polylCLC. Based on these results, three pools were selected. Two pools each with only one peptide in 0.25 mM SS in D5W and a third pool with 5 peptides in 5 mM SS in D5W. The pH of the pools after being combined with polylCLC were all pH 5.0-6.0 and there was minimal loss during filtration.
  • D5W was prepared by weighing the dextrose and adding milliQ water to the dextrose to reach the appropriate volume. For example, water was added to 12.5 g dextrose to reach a total volume of 250 mL.
  • the % peptide content of each peptide was determined as follows: The total theoretical TFA is equal to the sum of the number of positive charges (N-terminus, Arg, Lys, and His). That number was entered in to the following equation where MW is the molecular weight of the peptide:
  • Target gross weight (l3.2*l0000)/(%peptide content*%purity)
  • DMSO (pL) (Actual gross weight (mg)*264 pl)/(Target gross weight (mg))
  • the stock was then diluted to 2 mg/mL (1 part DMSO stock, 24 parts buffer) in the appropriate formulation buffer.
  • Peptides were further diluted 1 :4 with buffer to obtain 0.4 mg/mL or, only when indicated, was buffer added directly to the dry peptide to obtain 0.4 mg/mL. In the latter case, Equation 6 was used to determine the appropriate volume to add.
  • Buffer (ml) (Actual gross weight (mg)* 33 mL)/ (Target gross weight (mg))
  • peptides L7, L8, L9, L14, LlOc, Ll ld, Ll lf and L15 were selected for formulation studies. Formulations without DMSO were tested as way to improve stability of formulated peptides and to slow down dimerization of cysteine-containing peptides. All peptides tested (L7, L8, L9, L14, LlOc, Ll ld, Ll lf and L15) at a concentration of 0.4 mg/mL in 0.25 mM SS and were soluble without DMSO after 6 hours.
  • Peptides L7, L8, L9, LlOc, Ll2d and L14 were tested in 5 mM SS and were also soluble without DMSO after 6 hours.
  • the pH values of the peptide formulations in 0.25 mM SS/D5W and 5 mM SS/D5W are listed in Table 12.
  • pool 3 and every pool containing peptide LlOc precipitated when combined with polylCLC. Additionally, pools with 3 peptides had a pH below 5.0 when combined with polylCLC, which suggests that the buffering capacity should be higher when a pool contains more than two peptides.
  • L7, L8, L9, LlOc, and L14 are all soluble in 5 mM SS they were tested in pools with the high SS concentration. Three pools were tested with these five peptides. One pool was without LlOc, one had LlOc alone, and the third had all five peptides. Because Ll lf and L15 were not soluble in this higher concentration they were formulated in 0.25 mM SS. However, due to the observed precipitation they were formulated to 0.4 mg/mL separately rather than in a single pool. Each of the peptides was soluble in their respective formulations. These pools were also compatible with polylCLC based on visualization and the pH values after the pools were combined with polylCLC were all between 5.0 and 6.3 (Table 15), which is appropriate for subcutaneous injection.
  • pool 1 (L7, L8, L9, LlOc, and L14 in 5 mM SS/D5W), pool 2 (either Ll li or Ll lf in 0.25 mM SS/D5W), and pool 3 (L15 in 0.25 mM SS/D5W).
  • Each of these pools was tested for retention on a 0.2 pm filter from Pall (HP1002).
  • the pre-filtered sample as well as sample after each of 2 filtrations were analyzed by UPLC-MS. Less than 3% of Ll lf and Ll li was lost after the first filtration step and no additional peptide was lost after the second filtration step. Only 4.9% L15 was lost after the first filtration and then 1.3% was lost after the second filtration. Less than a total of 3% of each peptide in Pool 1 was lost after the two filtrations steps.
  • a series of potential GATA3 peptides were tested for solubility in the formulation buffer contain 5mM SS/D5W with 4% DMSO. Peptides that were insoluble in 5mM SS were also tested in lower SS concentrations. Based on these results seven peptides were selected with five of them being soluble in 5 mM SS (L7, L8, L9, LlOc, and L14) and the others being soluble in the lower concentration (L 11 f , Ll li, and L15). Removal of DMSO was also tested and may improve solubility and slow disulfide formation, which can make UPLC analysis more difficult. Each of the peptides selected was soluble without DMSO.
  • Peptides L15 and Ll lf or Ll li were kept as individual peptides to be prepared by dissolving them directly at 0.4 mg/mL with 0.25 mM SS/D5W. These pools were all above pH 5.0 when mixed with poly- ICLC, which is acceptable for subcutaneous injection.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dermatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des compositions comprenant un peptide à solubilité améliorée et leurs procédés d'obtention. Dans un mode de réalisation, la présente composition comprend au moins un peptide, un modificateur de pH présent à une concentration inférieure à 1,0 mM, et un excipient pharmaceutiquement acceptable. La composition peut en outre comprendre d'autres constituants tels qu'un adjuvant immunomodulateur. L'invention concerne également des procédés de préparation des présentes compositions et des méthodes de traitement d'un sujet par l'administration des présentes compositions.
PCT/US2019/028204 2018-04-19 2019-04-18 Formulations peptidiques et utilisations associées Ceased WO2019204663A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201862660027P 2018-04-19 2018-04-19
US62/660,027 2018-04-19

Publications (1)

Publication Number Publication Date
WO2019204663A1 true WO2019204663A1 (fr) 2019-10-24

Family

ID=68239935

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2019/028204 Ceased WO2019204663A1 (fr) 2018-04-19 2019-04-18 Formulations peptidiques et utilisations associées

Country Status (3)

Country Link
AR (1) AR115346A1 (fr)
TW (1) TW202011982A (fr)
WO (1) WO2019204663A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12370255B2 (en) 2017-04-04 2025-07-29 Barinthus Biotherapeutics North America, Inc. Peptide-based vaccines, methods of manufacturing, and uses thereof for inducing an immune response

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016100975A1 (fr) * 2014-12-19 2016-06-23 Massachsetts Institute Ot Technology Biomarqueurs moléculaires pour l'immunothérapie d'un cancer
WO2016201049A2 (fr) * 2015-06-09 2016-12-15 The Broad Institute Inc. Formulations de vaccins contre la néoplasie et méthodes de préparation de celles-ci
WO2018187515A1 (fr) * 2017-04-04 2018-10-11 Avidea Technologies, Inc. Vaccins à base de peptides, procédés de fabrication et utilisations de ceux-ci pour induire une réponse immunitaire

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016100975A1 (fr) * 2014-12-19 2016-06-23 Massachsetts Institute Ot Technology Biomarqueurs moléculaires pour l'immunothérapie d'un cancer
WO2016201049A2 (fr) * 2015-06-09 2016-12-15 The Broad Institute Inc. Formulations de vaccins contre la néoplasie et méthodes de préparation de celles-ci
WO2018187515A1 (fr) * 2017-04-04 2018-10-11 Avidea Technologies, Inc. Vaccins à base de peptides, procédés de fabrication et utilisations de ceux-ci pour induire une réponse immunitaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OTT, PA ET AL.: "An Immunogenic Personal Neoantigen Vaccine for Melanoma Patients", NATURE, vol. 547, no. 7662, 13 July 2017 (2017-07-13), pages 217 - 221, XP002785348 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12370255B2 (en) 2017-04-04 2025-07-29 Barinthus Biotherapeutics North America, Inc. Peptide-based vaccines, methods of manufacturing, and uses thereof for inducing an immune response

Also Published As

Publication number Publication date
TW202011982A (zh) 2020-04-01
AR115346A1 (es) 2020-12-23

Similar Documents

Publication Publication Date Title
JP7535489B2 (ja) 治療用抗癌ネオエピトープワクチン
JP7384884B2 (ja) 異なるタイプのがんに対する免疫療法で使用するための非標準起源のペプチドおよびペプチド組み合わせ
AU2020230295B2 (en) Formulations for neoplasia vaccines
JP7754971B2 (ja) ネオ抗原およびそれらの使用
JP2022191218A (ja) 前立腺がんおよびその他のがんに対する免疫療法において使用するための新規ペプチドおよびペプチドの組み合わせ
JP2023093483A (ja) 子宮がん治療法
KR102095670B1 (ko) 종양 특이적 신생항원을 확인하는 조성물 및 방법
CN109310739A (zh) 新抗原及其使用方法
KR20180016531A (ko) 신생물 백신을 위한 제형 및 그의 제조 방법
JP7712919B2 (ja) マルチドメインタンパク質ワクチン
ES2970223T3 (es) Péptidos
AU2019344565A1 (en) B*44 restricted peptides for use in immunotherapy against cancers and related methods
JP2024001045A (ja) Ｎｓｃｌｃ、ｓｃｌｃ、およびその他のがんをはじめとする肺がんに対する免疫療法で使用するための新規ペプチドおよびペプチド併用
JPWO2019246286A5 (fr)
AU2019270985A1 (en) Peptides for use in immunotherapy against cancers
EP4100051A2 (fr) Traitement de maladies liées au hpv
WO2019204663A1 (fr) Formulations peptidiques et utilisations associées
JP2020014460A (ja) 数種の血液腫瘍、特に慢性リンパ性白血病（ｃｌｌ）に対する新規免疫療法
JP2023030082A (ja) Ａｍｌおよびその他のがんに対する免疫療法において使用するための新規ペプチドおよびペプチドの組み合わせ
US11191828B2 (en) MHC class I associated hepatitis B peptides
RU2813924C2 (ru) Неоантигены и их применение
HK40086688A (zh) 新抗原及其使用方法
HK40050845A (en) Neoantigens and uses thereof
JP2008308474A (ja) 抗原ペプチド製剤の調製方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19787919

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19787919

Country of ref document: EP

Kind code of ref document: A1