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WO2013033271A2 - Méthode permettant d'améliorer le système immunitaire en réaction à une maladie ou une lésion - Google Patents

Méthode permettant d'améliorer le système immunitaire en réaction à une maladie ou une lésion Download PDF

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Publication number
WO2013033271A2
WO2013033271A2 PCT/US2012/052944 US2012052944W WO2013033271A2 WO 2013033271 A2 WO2013033271 A2 WO 2013033271A2 US 2012052944 W US2012052944 W US 2012052944W WO 2013033271 A2 WO2013033271 A2 WO 2013033271A2
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immune
patient
lymphocytes
blood sample
cells
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WO2013033271A3 (fr
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Derren Barken
Israel Barken
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Priority to US13/970,537 priority Critical patent/US20140065629A1/en
Priority to US13/971,847 priority patent/US20140065098A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2026—IL-4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/204—IL-6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2046—IL-7
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/428—Undefined tumor antigens, e.g. tumor lysate or antigens targeted by cells isolated from tumor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/58—Prostate

Definitions

  • the field of the present invention relates generally to treatments of disease in humans and animals.
  • the present invention relates to the identification and use of immunostimulatory reagents in such treatments.
  • the individual's immune system recognizes the presence of a "foreign body” that carries an antigen, which antigen is perceived as new or foreign by the individual's immune system even though the new antigen was made by the individual's own cells. That recognition is referred to as an "immune recognition event" in the description of the present invention herein below.
  • the immune system's reaction is to attack the bearer of the new antigen, which process includes an upsurge of a particular subset of T cells that share a common receptor.
  • the subset of T cells having the common receptor can be identified by being among the most abundant lymphocytes relative to the total population of lymphocytes in the individual's blood, and being a newly abundant such subset of T cells subsequent to the immune recognition event.
  • the newly abundant subset of such T cells is referred to as a "clonotype.”
  • Each T cell clonotype mounts an attack on a particular antigen that is perceived as foreign; the act of perceiving an antigen as foreign is what is referred to herein as an "immune recognition event" that triggers downstream effects.
  • immune recognition event that triggers downstream effects.
  • many such immune attacks remain subclinical, providing limited or no good effect to the individual. Nonetheless, the fact that an immune attack began by the individual's built-in system of protection is a signal of a path for ameliorating the particular disease or injury that is harming the individual. Accordingly, if the immune attack mounted by the immune system of an individual suffering from disease or injury is detected in a timely fashion, then a physician could be in a position to administer general and known immune-boosting agents at a time likely to have a good effect. Moreover, if the immune attack is analyzed to identify the path the individual's own immune system is taking, a physician could be in a position to order immuno-reagents specific for the particular patient and his, her, or its particular disease or injury.
  • the key is building methodologies for identifying when an individual suffering from disease or injury experiences an immune recognition event so that a general and/or personalized immune -boosting treatment can be timely administered for optimum effect. Further methodologies are needed for understanding greater detail of the immune recognition event so that reagents defined by that patient's own immune system can be identified, generated and administered.
  • Cancer is a large, heterogeneous class of diseases in which a group of cells display uncontrolled growth, invasion that intrudes upon and destroys adjacent tissues, and often metastasizes, wherein the tumor cells spread to other locations in the body via the lymphatic system or through the bloodstream.
  • These three malignant properties of cancer differentiate malignant tumors from benign tumors, which do not grow uncontrollably, directly invade locally, or metastasize to regional lymph nodes or distant body sites like brain, bone, liver, or other organs.
  • the present invention presents novel methods of analysis and treatment that provides protocols for timely increasing the general attacking power of an individual's immune system and for building efficacy into a quantity of an individual's native lymphocytes that are geared to attack the perceived foreign object as understood by the patient's own immune system.
  • One embodiment provides a method for identifying DNA or RNA sequences of lymphocyte receptors that are present in greater numbers of lymphocytes and/or are more highly expressed after a medical procedure, the method comprising drawing pre- procedure blood from a cancer patient, carrying out a medical procedure on the patient, drawing blood from the patient at one or more times following the medical procedure, purifying lymphocytes from any one or more of the pre-procedure blood draw(s), and/or the post-procedure blood draw(s); isolating DNA or mRNA or both, in the case of the DNA amplifying (if necessary) and sequencing to provide the cell copy number of the TCR gene and in the case of the mRNA assaying for quantity of TCR-encoding messenger to provide the level of expression; dividing RNA units of expression by DNA copy provides level of expression per cell that can be used in treatment. The most direct result of this effort is to identify lymphocytes and/or lymphocyte receptor sequences that have expanded following the medical procedure.
  • Another embodiment provides a method wherein the patient is selected based on the severity or extent of cancer (Gleason Score for example) and/or patient treatment status.
  • Another embodiment provides a method wherein the procedure comprises one or more
  • Another embodiment provides a method wherein the cancer is prostate cancer and the medical procedure comprises one or more of cryosurgery, radical prostatectomy, prostate biopsy, radiation therapy, brachytherapy, robotic-mediated radiotherapeutic procedures, electrop oration, high frequency ultrasound (HIFU), photodynamic therapy, prostate laser surgery, androgen deprivation therapy, and chemotherapy.
  • the medical procedure comprises one or more of cryosurgery, radical prostatectomy, prostate biopsy, radiation therapy, brachytherapy, robotic-mediated radiotherapeutic procedures, electrop oration, high frequency ultrasound (HIFU), photodynamic therapy, prostate laser surgery, androgen deprivation therapy, and chemotherapy.
  • Another embodiment provides a method wherein the procedure leads to an immunogenic response.
  • Another embodiment provides a method wherein the procedure results in a change in the population lymphocytes.
  • lymphocytes include Tcells displaying various TCRs.
  • Another embodiment provides a method wherein treatment of cancer comprises inducing in a patient an immunologic response incorporating clonotypes identified by the method of claim 6.
  • Another embodiment provides a method wherein the method further comprises selecting clonotypes as highly expanded if their frequency (in the measured repertoire) is 0.5% or greater.
  • Another embodiment provides a method wherein the method further comprises selecting a clonotype that is absent or not highly expanded prior to cryosurgery, but which is highly expanded after cryosurgery as a tumor associated clonotype.
  • Another embodiment provides a method wherein the method further comprises selecting a clonotype as a tumor specific clonotype if it is highly expanded both before and after a medical procedure, but has a frequency that increases from before to after the procedure, wherein the increase is statistically significant using an appropriate multiple hypothesis testing statistical method to stringently limit the false discovery rate.
  • Another embodiment provides a method wherein the method further comprises extracting tissue from the patient for use in an in vitro assay of autologous engineered T cells.
  • Another embodiment provides a method wherein the procedure comprises receptor chain pairing.
  • Another embodiment provides a method wherein chain pairing is carried out in silico by computer methods.
  • Another embodiment provides a method wherein chain pairing involves immunology gene alignment software.
  • Another embodiment provides a method wherein the software is selected from IMGT,
  • Another embodiment provides a method wherein chain pairing involves using VDJ antibodies.
  • Another embodiment provides a method wherein the method further comprises obtaining antibodies for the identified segments and use the antibodies to purify a subset of cells which express that gene segment in their (surface) receptors (e.g., using fluorescence-activated cell sorting (FACS) or immunomagnetic selection with microbeads).
  • FACS fluorescence-activated cell sorting
  • microbeads immunomagnetic selection with microbeads
  • Another embodiment provides a method wherein the method further comprises sequencing a subset of cells which have been purified for the desired gene segments.
  • Another embodiment provides a method wherein chain pairing is carried out using multiwell sequencing or single cell sequencing.
  • Another embodiment provides a method wherein the method further comprises genetic engineering of autologous T cells, acquired by leukapheresis, to display the TCR or CAR of the induced clonotype(s).
  • Another embodiment provides a method wherein a T cell is engineered to display a functional TCR.
  • Another embodiment provides a method wherein a chimeric cell is engineered in which a T cell displays an alternative type of receptor such as a chimeric antigen receptor.
  • Another embodiment provides a method wherein the method further comprises an in vitro assay.
  • Another embodiment provides a method wherein the engineered T cells are incubated with tumor tissue or lysate.
  • Another embodiment provides a method wherein one or more effects are measured during the incubation such as cytokine concentration, cell proliferation, and the like. In some embodiments, the effects of various adjuvants are quantified.
  • Another embodiment provides a method wherein the engineered T cells are provided as a treatment for cancer.
  • Figure 1 shows a flowchart corresponding to protocol for the study of in vitro and in vivo efficacy of autologous engineered T cells.
  • Figure 2 shows excerpts from a sample TCR profile report.
  • Figure 3 shows excerpts from a sample TCR profile report.
  • Figure 4 shows a scatterplot demonstrating the reproducibility of the method, provided by a TCR and BCR profiling vendor (Adaptive Biotechnologies Corporation, Seattle, WA).
  • the present invention is a method of treatment that harnesses a patient's own immune system to effect cure or amelioration of symptoms of disease or injury.
  • the patient that may be successfully treated is human or animal.
  • Diseases and conditions that can be addressed runs the gamut from a cancer to heart disease to an inflammation: Literally, to any disorder, condition, or injury in response to which the body's immune system ordinarily mounts an attack.
  • the immune system is the body's police, truly seeking to bring inner peace by seeking and destroying carriers of foreign antigens.
  • inventive methods set forth herein and in a sister application collect information from the police (i.e., the body's immune system as observed in the lymphocytes by one skilled in the art and knowledgeable of the present invention) and use that information to (a) time when to administer immunostimulating cytokines in one embodiment, (b) identify lymphocyte clonotypes that can be usefully employed to cure or ameliorate the disease or injury that gave rise to the identified clonotypes, and (c) generate autologous lymphocytes of the identified clonotypes for infusion into the patient for waging an immunotherapeutic attack to a clinically observable level.
  • police i.e., the body's immune system as observed in the lymphocytes by one skilled in the art and knowledgeable of the present invention
  • TCR T cell receptor
  • sequence data also provides quantitative data for assessing the relative abundance of lymphocytes in a blood sample having each identified TCR, which in turn tells us the most abundant clonotypes in the blood sample. Observing changes in the most abundant clonotypes in blood samples from before an event that induces an immune recognition event and after that event is used in one embodiment of the present invention. This observation of the body's police tells us which clonotypes are being assembled to handle the insult that proximately induced an immune recognition event (which is the event that results in creating new clonotype lymphocytes geared for addressing the insult).
  • a malignant tumor is itself commonly a silent insult that avoids the body's immune system.
  • disruption of the tumor results in revealing antigens that the immune system sees as foreign, albeit commonly a weak, clinically-limited immune response ensues.
  • a physician can assemble an efficacious culture of lymphocytes that have the TCR of the abundant clonotype elicited by the insult visited upon the tumor cells, which resulted in the immune recognition event and the generation of a clonotype that the inventive method showed was being martialed to address the tumor.
  • the present invention is amenable for augmenting the immune system reaction that occurs in the context of any disease or injury, it is worthwhile mentioning some of the diseases that can be addressed.
  • Parkinson's Disease associated with degeneration of dopamine neurons in the substantia nigra pars compacta part of the brain where the role of inflammation mediates the innate and adaptive immune systems.
  • Panaro and Cianciulli “Current Opinion and Perspectives on the Role of Immune System in the Pathogenesis of Parkinson's Disease," Curr. Pharm. Des. 18(2):200-8 (2012).
  • the present inventive method is also amenable for addressing interstitial cystitis and periodontal disease.
  • that effort is boosted generally by administering suitable cytokines in suitable combinations at a time point that is proximate to the occurrence of an action that results in an immune recognition event.
  • that effort is boosted specifically by administering to a patient a heightened concentration of autologous lymphocytes that share the TCR or an optimized engineered form thereof of the more abundant lymphocyte clonotypes that arose in the patient's blood subsequent to the immune recognition event.
  • the present invention comprises a medical procedure, and sequencing of the T cell repertoire before and after a medical procedure, in order to detect and sequence the TCR alpha and beta loci of highly expanded T cell clonotypes found in the blood.
  • An important and very helpful variation on selection of source of lymphocytes to be studied in the context of the inventive method is to assess the TCR and BCR repertoire changes before and after an immune recognition event in the bone marrow, which is readily accessed using standard bone marrow aspirations.
  • the differential information relating to the receptor abundancies among lymphocytes is used to create autologous genetically engineered T cells and/or B cells with lymphocyte receptors that target the individual's tumor. Additionally, engineered recepters can also be introduced to the autologous cells and infused into the patient.
  • lymphocytic repertoire is being transformed by the advent of high throughput sequencing technologies.
  • the lymphocyte repertoire was widely studied using techniques such as spectratyping.
  • spectratyping To assess mixtures of lymphocytes from a patient's blood that include clonotypes that surged in abundance in consequence of the immune recognition event.
  • Such a mixture can be expanded in culture and used to stimulate the patient's immune system without having to sequence any DNA and without having to create an engineered autologous lymphocyte.
  • Lymphocytes have been attractive targets for these new techniques, for a variety of reasons.
  • the described methods of treatment incorporate a medical procedure which results in an in situ insult to an individual's tumor (such as cryosurgery).
  • the methods described herein comprise techniques for analyzing an individual's repertoire of lymphocyte receptors. Also described herein are methods that involve extracting lymphocytes, manipulating them ex vivo for therapeutic purposes, and then returning those lymphocytes to the individual to induce a therapeutic result.
  • One embodiment of the present invention describes a technique by which a medical procedure (also termed a tumor insult) may be employed to elicit an immunological response.
  • This response may be analyzed in detail by receptor repertoire sequencing of lymphocytes. The analysis may be expected to reveal the receptor sequences of lymphocyte clonotypes which are specific to the cancer. These sequences may be used to genetically engineer lymphocytes which have the same or similar tumor specificity but which may be manipulated ex vivo to enhance their anti-tumor efficacy when returned to the body as an immunotherapy.
  • the invention combines an in situ insult that induces an immune recognition event, such as freezing, irradiating, or biopsying a tumor, a series of one or more measurements of the T and/or B cell receptor repertoire, an analysis of the T and/or B cell repertoire measurements in order to identify specific T and/or B cell receptor sequences expressed in specific T and/or B cell clonotypes that surged in abundance subsequent to the immune recognition event, and T and/or B cell gene therapy techniques to employ the identified TCR and/or BCR sequences for therapeutic purposes.
  • an immune recognition event such as freezing, irradiating, or biopsying a tumor
  • T and/or B cell receptor repertoire such as freezing, irradiating, or biopsying a tumor
  • an analysis of the T and/or B cell repertoire measurements in order to identify specific T and/or B cell receptor sequences expressed in specific T and/or B cell clonotypes that surged in abundance subsequent to the immune recognition event
  • T and/or B cell gene therapy techniques to employ the identified TCR and/or
  • Autologous T and/or B cells can be engineered using well-established protocols set forth elsewhere herein and well-known to a skilled artisan to express the identified TCR and/or BCR and/or CAR therein; the autologous T and/or B cells can be expanded in culture, again using protocols that are tried and true in the art; and the engineered autologous T and/or B cells once expanded can then be infused or otherwise administered into the patient whose cells they were in the first place.
  • the invention provides methods for generating an insult to a tumor, thereby inducing an immune recognition event and provoking an immune response which may be measured.
  • the invention provides methods for measuring said immune response in such a way as to be useful for the design of a gene therapy which is efficacious against a patient's tumor.
  • the invention provides methods for generating a gene therapy and/or immunotherapy which utilizes the information which is made available by an analysis of sequencing data sets which describe a T cell and/or B cell receptor repertoire.
  • the invention provides a description of the overall design which combines the individual elements described above into a multi-step clinical strategy which is efficacious.
  • this figure describes a method for determining T cell receptors induced or expanded by tumor intervention.
  • the figure presents a protocol that can be employed with respect to any disease or injury whose onset or subsequent treatment results in an immune recognition event.
  • the protocol of the present invention in the context of treatment of a patient afflicted with a tumor, this description is absolutely not intended to be limiting to a tumor treatment only, or to a disease treatment only for that matter, but is usefully considered as description of one embodiment of the inventive method that can be analogously employed with regard to any disease or injury that induces an immune recognition event or that, upon treatment thereof, induces an immune recognition event.
  • the protocol presents a method to identify the response of the patient's own immune system and then augment that line of attack to achieve a clinical effect resulting in cure or amelioration of symptoms.
  • lymphocyte purification step is optional because tools are available that allows the following steps to proceed without difficulty, as is known in the art.
  • Other steps can vary as well, as one can perceive in viewing the structure of our claims below, where fewer than all of the steps set forth in Figure 1 are included in our broader claims, which claims as recited describe a completely operative invention.
  • Figure 1 describes an in vitro (steps 1-8) and in vivo (step 9) study.
  • Patients may be selected for the study based on the severity or extent of their cancer (Gleason Score for example), their hormonal treatment status (androgen deprivation therapy, for example), and the like.
  • a population of patients are selected for study that statistically represent the patient population as a whole and/or a subset of the patient population suitable for treatment using the methods described herein.
  • one or more pre-operative blood draws are taken from a patient afflicted with cancerous tissue. These blood draws may be analyzed immediately or preserved for later analysis using any suitable method known to the art that preserves the integrity of the contained cells so that a later access of the contained genomic DNA can be subjected to DNA sequencing and analysis.
  • the cancerous tumor and/or cancerous tissue of the patient(s) are "insulted” or “intervened” or “treated,” meaning that the tissue is acted upon, treated, surgically altered, altered by radiation or other nonsurgical intervention, or exposure to a drug and the like.
  • Any medical procedure known in the art of cancer treatment is appropriate, and may include various types of prostate surgery in various embodiments. Non- limiting examples include one or more of cryosurgery, radical prostatectomy, prostate biopsy, radiation therapy, brachytherapy, robotic-mediated radiotherapeutic procedures, electrop oration, high frequency ultrasound (HIFU), photodynamic therapy, prostate laser surgery, androgen deprivation therapy, and chemotherapy.
  • a method of tumor insult is selected that is known to lead to an immunogenic response. Without being limited to any particular theory, it is
  • the lymphocytes include T-cells displaying various T cell receptors (TCRs).
  • TCRs T cell receptors
  • TCRs that are specific to the cancerous tumor can be utilized in methods for treatment of the cancer.
  • the cancerous tissue is analyzed immediately or preserved for later analysis. Methods of analysis can include methods described herein or any suitable method of genetic and/or biochemical analysis known to those skilled in the art.
  • the tissues are preserved, optionally as formalin-fixed, paraffin embedded (FFPE) tissue.
  • FFPE formalin-fixed, paraffin embedded
  • blood is drawn from the patient at various times following intervention of the cancerous tissue. Any time period may be suitable and may be adjusted to coincide with the timing of an immunological response in the patient.
  • blood is drawn at a plurality of times.
  • blood is drawn on the same day as the intervention, and again at 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 30 days, and the like following surgery.
  • the post-operative and/or post-intervention blood samples may be analyzed immediately or preserved for later analysis.
  • lymphocytes are separated and purified from the drawn blood.
  • One example method of lymphocyte separation comprises layering heparinized venous blood onto a density gradient of Ficoll-Isopaque (GE Healthcare Biosciences, Pittsburgh, PA) in 15 ml conical tube in the ratio 3: 1 ; centrifuging the tube at about 1800 rpm for about 20 minutes; removing the middle layer;
  • RPMI- 1640 medium Life Technologies Corporation, Grand Island, NY
  • fluorescent reagents such as monoclonal antibodies specific for CD4 or CD8 cells, which antibodies are tagged with a fluorochrome, such as phycoerythrm (PE) or 5(6)-fluorescem isothiocyanate (FTTC) (BD Biosciences Pharmingen, San Diego, CA); vortexing the suspension at room temperature; incubating the suspension for 2 hours in the dark at room temperature; and identifying the specific lymphocytes based on staining with specific antibodies for sorting on a cell sorter apparatus such as the BD FACStar Plus (BD Biosciences, San Jose, CA
  • Separation and purification can also be accomplished by immunomagnetic selection with microbeads.
  • the immunomagnetic selection can be done with commercially available kits such as Miltenyi CD8+ kits (Miltenyi Biotec Inc., Auburn, CA).
  • Other separation methods such as fluorescence- activated cell sorting (FACS) can also be used to separate and purify lymphocytes from the drawn blood. Lymphocyte isolation may be done using positive isolation, negative isolation, or a sequence of steps that includes both positive and negative isolation.
  • CD3+ T Cells can be separated using an Invitrogen Dynabeads CD3 kit (Catalog #1 1 151D or 1 1365D, available from Life Technologies
  • kits for isolation of other lymphocytes; other suitable separation methods can also be used.
  • a Pluriselect kit Catalog #10-00300- 21 , Pluriselect GmbH, Leipzig, Germany
  • DNA is isolated from any one or more of the pre-operative blood draw(s), the cancerous tissue, and/or the post-operative blood draw(s).
  • DNA is extracted from a mixed tissue or mixed cell-type sample, optionally from whole blood or cancerous tissue. This embodiment may eliminate the need for certain sample processing steps, whereby the genetic loci of interest can be interrogated from a mixed DNA sample.
  • the blood and/or tissue samples are first enriched for certain lymphocytes, optionally by whole blood fractionation. Whether from an enriched sample, or from a non-enriched sample, DNA can be isolated according to any suitable method known to those skilled in the art.
  • kits such as QiA DNA Blood Maxi prep #51 192 or QIAGEN DNeasy Blood and Tissue kit #69506.
  • CDNA may also be prepared with kits such as QIAGEN FastLane Cell cDNA Kit #21501 1. The identified kits are available commercially from QIAGEN Inc. (Sunnyvale, CA).
  • step 4 of Figure 1 the extracted DNA is then amplified (if necessary) and sequenced.
  • the DNA encoding the lymphocyte receptors is amplified in some embodiments, optionally the T cell receptors.
  • T cell receptors consist of alpha (a) and beta ( ⁇ ) chains.
  • both the alpha and beta chains of a TCR are amplified.
  • Primers are short strands of DNA that physically stick (or anneal) to the ends of the DNA, and allow other molecules (known as polymerases) to "copy" what is in between them.
  • various loci can be amplified separately.
  • the alpha and beta chains of a TCR can be amplified separately to yield two PCR products.
  • PCR polymerase chain reaction
  • Step 4 of Figure 1 includes DNA sequencing of the DNA.
  • Methods of DNA sequencing are well known in the art and have improved rapidly in recent years in features such as read length, improved throughput, reduced cost and the like.
  • One suitable method of DNA sequencing is pyrosequencing.
  • Pyrosequencing is a method of DNA sequencing (determining the order of nucleotides in DNA) based on the "sequencing by synthesis" principle. It differs from Sanger sequencing, in that it relies on the detection of pyrophosphate release on nucleotide incorporation, rather than chain termination with dideoxynucleotides as used in the Sanger method.
  • DNA sequencing results in sequence data of adenine (A), cytosine (C), thymine (T) and guanine (G) that is analyzed by computer methods. Sequencing may be done by vendors such as Adaptive Biotechnologies Corporation (Seattle, WA).
  • Step 5 includes methods for identifying lymphocytes and/or lymphocyte receptor sequences that have expanded following intervention of the cancerous tissue. For most locations along the DNA, the sequence is identical from cell to cell. However, the T cell receptor and B cell receptor locations are different. DNA regions coding for T cell receptor and B cell receptor, the body uses a process known as somatic or "VDJ" recombination to recombine the DNA at that location (in one individual cell). Therefore, if one takes a population of T cells or B cells (from a blood draw), and carries out a targeted resequencing of the DNA at the T cell or B cell receptor locus, one will get many different "reads" - or sequences of DNA (each read comes from a different cell).
  • VDJ somatic or
  • a mixture of primers which includes a plurality of primers. This provides for "multiplexed” PCR.
  • the primers correspond to the DNA at either end of the DNA targeted for amplification. In this way, the primers act as "bookends.”
  • the bookends vary from cell to cell, but the possible bookends come from a small, fixed set.
  • V segments and C (constant) segments there are known bookends called V segments and C (constant) segments and because the resequencing is done using a mixture of primers, all the possibilities are covered. Every T cell receptor or B cell receptor will include one V segment and one C segment.
  • the DNA between the bookends is amplified and the amplified DNA may then be sequenced using well known techniques such as pyrosequencing. These sequences represent a "repertoire" which can be analyzed. In particular, one may decipher which sequences are more or less common. In addition, one may take two blood draws ("before” and "after") and decipher which sequences were absent before a medical procedure but appeared after the medical procedure.
  • Expansion it is meant that the number of members of a clonotype is greater relative to other clonotypes found in the same blood sample following intervention or injurious event that induces an immune-recognition event; as compared to the clonotypes identified in a blood sample collected from the same patient before intervention or injurious event. Expansion can be quantified by comparing the amount of amplified DNA for a given alpha and/or beta chain from samples before and after intervention or injurious event.
  • Step 5 The methods of Step 5 are often computer-based.
  • clonotypes are considered highly expanded if their frequency (in the measured repertoire) is 0.5% or greater.
  • a clonotype which is absent or not highly expanded prior to cryosurgery or other medical procedure, but which is highly expanded after cryosurgery or other medical procedure is inferred to be a tumor associated clonotype.
  • a clonotype will be inferred to be a tumor specific clonotype if it is highly expanded both before and after tumor intervention, but has a frequency that increases from before to after cryosurgery or other medical procedure, where the increase is statistically significant using an appropriate multiple hypothesis testing statistical method to stringently limit the false discovery rate.
  • sequencing is replaced with spetratyping (also known as immunoscoping).
  • spetratyping also known as immunoscoping.
  • Spectratype analysis takes advantage of PCR technology to amplify template cDNA corresponding to rearranged transcripts with different complementarity determining region 3 (CDR3) lengths from specific TCR variable region genes in a competitive manner.
  • CDR3 complementarity determining region 3
  • the spectratype analysis of a patient's lymphocytes is carried out before and after an immune recognition event, such as a medical procedure intervention or injury event, to identify the particular lymphocyte receptor V and/or J segments that expanded subsequent to the immune recognition event.
  • an immune recognition event such as a medical procedure intervention or injury event
  • a subset of cells expressing the identified V and/or J segments can then be isolated from the patient's own blood, expanded, stimulated, or modified ex vivo, and then reinfused as a therapeutic treatment.
  • Figure 1 is a depiction of generalized procedures and not limiting.
  • Step 4 of Figure 1 does not limit the method to performing DNA extraction, amplification and sequencing simultaneously.
  • steps not necessarily depicted in the figures such as sample processing and the like; and the listing of steps in a given order is not in and of itself a recital that the identified steps must be accomplished in that order, irrespective of how they are listed or described in the detailed description, figures, examples, or, for that matter, claims.
  • steps 4 and 5 comprising the steps of: purification; DNA isolation; amplification; sequencing; and identification of lymphocyte clonotypes may be carried out immediately after each blood draw or blood samples drawn before an immune recognition event can be stored and processed per steps 4 and 5 at the same time that a blood sample is drawn subsequent to the immune recognition event.
  • Step 5 of Figure 1 results in separate data comprising alpha chains that are induced upon intervention of the tumor and beta chains that are induced upon intervention of the tumor.
  • Step 6 of the procedure depicted in Figure 1 involves "paired chain analysis".
  • various methods can be utilized to pair induced alpha and beta chains such that the pairing results in a TCR that binds to an epitope of the cancerous tissue or otherwise leads to an immune response targeting the cancerous tissue.
  • post- sequencing pairing may be unnecessary or relatively simple, for example in embodiments in which the alpha and beta chain pairing information is not lost in the procedure, such as if one were to sequence from single cells.
  • the chain pairing may be assisted in silico by computer methods for annotating VDJ gene segments.
  • IMGT International IMmunoGeneTics information system
  • the chain pairing may be done using VDJ antibodies. For example, one may obtain antibodies for the identified segments and use the antibodies to purify a subset of cells which express that gene segment in their (surface) receptors (e.g., using FACS, or immunomagnetic selection with microbeads). One may then sequence from this subset of cells which have been purified for the desired gene segments. If necessary, this secondary sequencing may be done more deeply (i.e. at a higher resolution) than the first round of sequencing. In this second sequence data set, there will be far fewer induced clonotypes, greatly easing the task of chain pairing. Depending on the gene segments, there may be only one induced alpha chain and one induced beta chain for example.
  • the chain pairing may be done by trial and error.
  • the chain pairing may be done using multiwall sequencing. For example, one may isolate gene segment purified cells or unpurified cells into a microwell plate, where each microwell has a very low number of cells. One can amplify and sequence the cells in each well individually, which provides another means to pair the chains of interest by sequencing on a single cell basis, facilitating the pairing of induced alpha and beta chains.
  • lymphocytes may be performed after each blood draw or may be performed after the last blood draw has been completed.
  • identification of lymphocyte clonotypes may also follow after each blood draw or after the completion of the intended blood draws with respect to the analysis surrounding the immune recognition event.
  • Step 7 of Figure 1 includes genetic engineering of autologous T-cells to display the TCR or chimeric antibody receptor corresponding to the induced clonotype(s).
  • Methods of genetic engineering are generally known in the art and can be found in well-known texts of protocols including Sambrook et al. (2001), MOLECULAR CLONING. A LABORATORY MANUAL, Cold Spring Harbor Press, Plainview, N.Y.), to provide one non-limiting example.
  • the alpha and beta chains of the T-cells of this invention may be expressed independently in different hosts or in the same host. Preferably the alpha and beta chains are introduced into the same host to allow for formation of a functional T-cell receptor in the host cell.
  • the host cell is capable of inducing an immune response in a patient.
  • the means by which the vector carrying the gene may be introduced into the cell include, but are not limited to, microinjection, electrop oration, transduction, retroviral transduction or transfection using DEAE-dextran, lipofection, calcium phosphate, particle bombardment mediated gene transfer or direct injection of nucleic acid sequences encoding the T cell receptors of this invention or other procedures known to one skilled in the art and set forth in well-known and utilized sourcebooks such as Sambrook et al., Id.
  • a T-cell is engineered to display a functional TCR.
  • a chimeric cell may be engineered in which a T-cell displays an alternative type of receptor such as a B-cell receptor.
  • Step 8 of Figure 1 includes in vitro assays.
  • the autologous engineered T- cells from Step 7 of Figure 1 are incubated with tumor tissue or lysate.
  • various effects are measured during the incubation such as cytokine concentration, cell proliferation, and the like.
  • the effects of various adjuvants are quantified.
  • T cell receptors may be applied in a treatment for cancer using T cell autologous or allogenic adoptive transfer cell therapy.
  • T cells from the patient
  • T cell receptor is suppressed, and an alternative T cell receptor sequence (as determined previously) is introduced into the cell.
  • T cell receptor sequence as determined previously
  • the cells further include one or more adjuvants suitable to illicit or amplify an immune response.
  • step 9 the effects of the treatment are evaluated by reference to clinical and surrogate endpoints.
  • the clinical endpoints may include one or more of overall survival, progression free survival, or tumor regression.
  • the surrogate endpoints may include longitudinal measurements of cancer biomarkers. In the case of prostate cancer, available surrogate endpoints would include PSA (prostate specific antigen) and circulating tumor cells.
  • FIGS 2 and 3 show that the TCR beta chain complementarity determining region 3 (CDR3) sequences and clonotype frequencies are commercially available.
  • the commercial provider is Evrogen Lab (Moscow, Russia).
  • Figure 2 is an exemplary report showing among other things, a listing of clonotypes (clones), their sequence and read count, the percentage of the clone in the V gene family, the percentage of the clonotypes in the J gene family.
  • Figure 3 the abundance of T-cell receptor V beta genes depicted as a histogram and a pie chart.
  • Figures 2 and 3 are examples of computer-based methods that can be used to identify T-cell receptor segments that increase in abundance following insulting the tumor.
  • Figure 4 shows a scatterplot demonstrating the reproducibility of the method.
  • both the x-axis and y-axis are the same logarithmic scale plotting the same data, so perfectly reproducible data would fall on a 45 degree line ascending from the bottom left to the top right of the graph.
  • Figure 4 shows that a majority of the data points follow this 45 degree trend of reproducibility.
  • the methods disclosed herein can be used to treat all types of cancer including but not limited to breast cancer, colon cancer, liver cancer and the like.
  • Either the primary or secondary tumors can be insulted.
  • lymph material is removed or drawn from the patient in lieu of blood.
  • One embodiment provides a method for identifying the DNA or RNA sequences of lymphocyte receptors expressed by lymphocytes that are present in increased numbers in a particular patient after a medical procedure; the method comprising: (i) drawing blood at one or more times from a cancer patient prior to a medical procedure (ii) carrying out a medical procedure; (iii) drawing blood from the patient at one or more times following the medical procedure; (iv) purifying a lymphocyte subpopulation, isolating DNA, amplifying (if necessary) and sequencing the genetic loci of receptors; and (v) identifying lymphocytes and/or lymphocyte receptor sequences that have expanded in number following the medical procedure.
  • Another embodiment provides a method wherein the analysis of lymphocyte receptor repertoire sequences, from patient samples obtained both before and after a medical procedure, enables the production of autologous genetically engineered T cells having transgenic T cell receptors which are specific to the patient's cancer.
  • Another embodiment provides a method wherein the analysis of lymphocyte receptor repertoire sequences, from patient samples obtained both before and after a medical procedure, enables the production of autologous genetically engineered T cells having chimeric antigen receptors which are specific to the patient's cancer.
  • Another embodiment provides a method wherein autologous engineered T cells, having transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs), are shown to be efficacious in vitro in mounting a cytotoxic and/or therapeutic immune response to the presence of tumor tissue or lysate.
  • TCRs transgenic T cell receptors
  • CARs chimeric antigen receptors
  • Another embodiment provides a method wherein autologous engineered T cells, having transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs), are shown to be efficacious in vivo in affecting clinical endpoints such as overall survival, progression free survival, or tumor regression.
  • TCRs transgenic T cell receptors
  • CARs chimeric antigen receptors
  • Another embodiment provides a method wherein autologous engineered T cells, having transgenic T cell receptors (TCRs) or chimeric antigen receptors (CARs), are shown to be efficacious in vivo in affecting surrogate endpoints such as longitudinal measurements of biomarker levels or circulating tumor cells.
  • TCRs transgenic T cell receptors
  • CARs chimeric antigen receptors
  • Another embodiment provides a method wherein the patient is selected based on the severity or extent of cancer (for example, a Gleason score in the context of prostate cancer), patient treatment status (for example, androgen deprivation therapy status in the context of prostate cancer), or other clinical status.
  • a Gleason score in the context of prostate cancer
  • patient treatment status for example, androgen deprivation therapy status in the context of prostate cancer
  • Another embodiment provides a method wherein the medical procedure, comprises one or more surgical procedures, nonsurgical interventions or pharmaceutical treatments.
  • Another embodiment provides a method wherein the cancer is prostate cancer and the medical procedure comprises one or more of cryosurgery, radical prostatectomy, prostate biopsy, radiation therapy, brachytherapy, robotic-mediated radiotherapeutic procedures, electrop oration, high frequency ultrasound (HIFU), photodynamic therapy, prostate laser surgery, androgen deprivation therapy, and chemotherapy.
  • the medical procedure comprises one or more of cryosurgery, radical prostatectomy, prostate biopsy, radiation therapy, brachytherapy, robotic-mediated radiotherapeutic procedures, electrop oration, high frequency ultrasound (HIFU), photodynamic therapy, prostate laser surgery, androgen deprivation therapy, and chemotherapy.
  • Another embodiment provides a method wherein the intervention leads to an immunogenic response.
  • Another embodiment of the present invention includes an onset of disease or injury or an intervention/treatment thereof, where a change in the population or repertoire of lymphocytes is observed and used to generate immunostimulating reagents.
  • the immunostimulating reagent in one such
  • the immunostimulating reagent can be lymphocytes having a TCR or CAR consistent with one of the identified newly abundant clonotypes identified in accordance with the present invention.
  • lymphocytes include B cells displaying various B Cell Receptors (BCRs).
  • Another embodiment provides a method further comprising selecting clonotypes as highly expanded or newly abundant if their frequency (in the measured repertoire) is 0.5% or greater.
  • Another embodiment provides a method further comprising selecting a clonotype that is absent or not highly expanded prior to a medical procedure, but which is highly expanded or abundant relative to other clonotypes in the patient's blood sample drawn after a medical procedure, which selected clonotype is referred to as a treatment-associated clonotype.
  • Another embodiment provides a method further comprising selecting a clonotype as a tumor specific clonotype if it is highly expanded both before and after a medical procedure, but has a
  • Another embodiment provides a method wherein the sequences of two chains comprising a lymphocyte receptor (e.g., alpha and beta TCR, gamma and delta TCR, or heavy chain and light chain BCR) are paired.
  • a lymphocyte receptor e.g., alpha and beta TCR, gamma and delta TCR, or heavy chain and light chain BCR
  • the chain pairing is carried out in silico by computer methods, or by use of immunology gene alignment software, or by using VDJ antibodies in accordance with knowledge generally known in the art, or by using multiwell sequencing as known in the art, or by manual means for assessing the DNA sequences of expected chain pairs as know in the art; wherein, in one embodiment, the software is selected from IMGT, JOINSOLVER, VDJSolver, SoDA, iHMMune-align, or other similar software tools.
  • Another embodiment provides a method comprising obtaining antibodies for the identified VDJ gene segments and using the antibodies to purify a subset of cells which express that gene segment in their (surface) receptors (e.g., using FACS, or immunomagnetic selection with microbeads).
  • Another embodiment provides a method further comprising sequencing a subset of cells which have been purified for the desired VDJ gene segments.
  • Another embodiment provides a method further comprising genetic engineering of autologous T-cells to display the TCR of the induced clonotype(s), or to display a functional TCR, or to display an alternative type of receptor, such as a B-cell receptor.
  • the engineered autologous T cells are incubated with tumor tissue and/or lysate of tumor tissue prior to administering same to the patient by infusion or other in vivo treatment.
  • One or more criteria are commonly measured during the incubation, such as cytokine concentration, cell proliferation, and the like.
  • the effects of various adjuvants are quantified.
  • the clinical benefit of treatment with engineered autologous T cells is measured in terms of surrogate endpoints such as longitudinal measurements of biomarker levels or circulating tumor cells.
  • lymphocytes bearing particular V and/or J segments are filtered and isolated in a filtration and purification step and incubated for autologic or allogenic cell transfer therapy without genetically engineering lymphocytes to express specific receptors on the cell surface.
  • spectratyping is carried out on one or more of T cell receptor alpha chains, beta chains, gamma chains, or delta chains; or, in the same fashion, B cell chains can be the subject of the
  • spectratyping As a result of spectratyping, a change in amount or composition (ologiclonality) of one or more V or J segments in one or more chains is identified if it exists in the differences between the before and after immune recognition event blood samples.
  • TRAV8-2, TRAJ4, TRBV17, and/or TRBJ2-5 a qualitative or quantitative increase in amount and oligoclonality may be seen in segments TRAV8-2, TRAJ4, TRBV17, and/or TRBJ2-5 (as defined in the book The T Cell Receptor FactsBook, by Marie-Paule Lefranc and Gerard Lefranc). Consequently, using appropriate commercial antibodies which are specific to the V and/or J segments identified, cells bearing receptors containing these segments are isolated by a standard and known method for sorting cells, such as fluorescence activated cell sorting (FACS), magnetic beads (Life Technologies dynabeads), or related methods (pluriSelect GmbH cell isolation technologies, to name another).
  • FACS fluorescence activated cell sorting
  • magnetic beads Life Technologies dynabeads
  • related methods pluriSelect GmbH cell isolation technologies, to name another).
  • subcompartment of the overall T cell or B cell repertoire.
  • the isolated subcompartment or subpopulation of T cells may then be manipulated or activated in vivo using a number of methods which will be known to one skilled in the art, including cytokines, anti-CD3/anti-CD28 antibodies (Catalog ##1 1 1-3 ID, 1 11-32D, 1 1 1-61 D; Life Technologies Corporation, Grand Island, NY), etc. These methods are used, for example, in T cell proliferation assays.
  • the relevant subcompartment or subpopulation of cells may be therapeutically reinfused. Their persistence and status may also be monitored post-infusion, with subsequent blood draws, as will be known to one skilled in the art. It is notable that this particular embodiment is relatively rapid and cost-effective, since spectratyping may be used instead of sequencing, and isolation using V and J specific antibodies may be used instead of genetic engineering. Further, by avoiding genetic engineering, this embodiment avoids problems with unwanted chain pairings.
  • both spectratyping and sequencing may be done, where spectratyping reveals the V and J segments of interest, and sequencing is done using only primers which correspond to the V and J segments of interest. This approach greatly reduces the amount of sequencing necessary, and, as well, provides the opportunity to utilize more rapid sequencing technologies.
  • a "panning experiment” is carried out after identifying expanded receptors before and after sequencing in which many copies of the identified receptor are incubated with a combinatorial library of possible epitopes. Once the corresponding epitope is identified, tetramers loaded with the identified epitope are used to isolate lymphocytes from the patient's own cells which are specific to that epitope. These cells can then be expanded ex vivo, manipulated, and then therapeutically infused.
  • One skilled in the art is familiar with the procedure (Li Pira et al., "High Throughput T Epitope Mapping and Vaccine Development," J. BlOMED. BlOTEHNOL. 2010:325720 (2010); Sung et al., "T-cell Epitope Prediction with Combinatorial Peptide Libraries," J. COMPUT. BIOL. 90 ⁇ :527-39 (2002).
  • cryotherapy is the local or general use of low temperatures in medical therapy or the removal of heat from a body part.
  • cryoablation is a process that uses cold (cryo) to destroy tissue (ablation). In cryoablation, the destroyed tissue typically remains in the body, enhancing an immune response.
  • a "lymphocyte” is a type of white blood cell in the vertebrate immune system that is used herein to refer to both T cell and B cell varieties of lymphocytes, either of which are amenable to the methods of the present invention; moreover, protocols described with reference to either a T cell or a B cell are equally available for application to the other lymphocyte variety.
  • T cells and B cells are called “small” lymphocytes, and “large” lymphocytes include macrophages and granular lymphocytes, such as natural killer cells (NK cells).
  • lymphocyte For the purposes of clarity in the specification of our invention, we refer to the small lymphocytes only when we recite the term "lymphocyte” herein; and when we intend to refer to a large lymphocyte as that term is understand in the art, we will recite the name of the particular cell.
  • T lymphocytes or "T cells” belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells (NK cells) by the presence of a special receptor on their cell surface called a T cell receptor (TCR), of which there are many, however molecular tools exist to identify the presence of differing TCRs by DNA sequence analysis of the genomic DNA from the
  • lymphocytes are lymphocytes.
  • a “cluster of differentiation” (often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules present on white blood cells, providing targets for CD.
  • CD molecules can act in numerous ways, often acting as receptors or ligands (the molecule that activates a receptor) important to the cell. A signal cascade is usually initiated, altering the behavior of the cell (see cell signaling). Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion. The number of CD proteins for humans is numbered up to 350 most recently (as of 2009).
  • CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). Like the TCR, CD8 binds to a major histocompatibility complex (MHC) molecule, but is specific for the class I MHC protein. There are two isoforms of the protein, alpha and beta, each encoded by a different gene. In humans, both genes are located on chromosome 2 at position 2pl2.
  • MHC major histocompatibility complex
  • T cell receptor or "TCR” is a molecule found on the surface of T lymphocytes (or T cells) that is, in general, responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the binding between TCR and antigen is of relatively low affinity and is degenerate: that is, many TCR recognize the same antigen and many antigens are recognized by the same TCR.
  • the TCR is composed of two different protein chains (that is, it is a heterodimer). In 95% of T cells in peripheral blood, this consists of an alpha (a) and beta ( ⁇ ) chain, whereas in 5% of T cells in peripheral blood, this consists of gamma and delta ( ⁇ / ⁇ ) chains. These percentages are different in other locations in the body.
  • VDJ recombination is also known as somatic recombination, is a mechanism of genetic recombination in the early stages of antibody (also referred to as immunoglobulin (Ig)) and T cell receptor (TCR) production of the immune system.
  • Antibodies and TCRs have structures in common that are designed to bind to antigens, which are components of any matter that are capable of inducing an immune reaction. Both antibodies and TCRs are formed, in part, by what is termed in the art as V(D)J recombination, which refers to the Variable, Diverse, and Joining gene segments found in vertebrates, of which there are a plurality of variant copies of each.
  • V(D)J gene segments are employed by an individual's immune system to create numerous different binding structures for portions of different antigens, generated and employed as and when the immune system is challenged by a new antigen.
  • the great variety of such binding structures needed for the many, many different possible antigens is generated by a nearly-random mechanism that selects one variety of the V gene segment and one variety of the J gene segment, marries them to a selected variety of the D gene segment, thereby generated DNA that encodes diverse but related proteins to match antigens from bacteria, viruses, parasites, dysfunctional cells (such as tumor cells or the consequences of inflammatory processes), pollens, and many, many more.
  • the "Gleason Grading system” is used to help evaluate the prognosis of men with prostate cancer. Together with other parameters, it is incorporated into a strategy of prostate cancer staging which predicts prognosis and helps guide therapy. A Gleason score is given to prostate cancer based upon its microscopic appearance. Cancers with a higher Gleason score are more aggressive and have a worse prognosis.
  • Robot-mediated radiotherapeutic procedures can be accomplished readily using a commercially available robotic system that is marketed under the brand name "CyberKnife” by Accuray Incorporated, Sunnyvale, CA.
  • the CyberKnife system is used for treating benign tumors, malignant tumors and other medical conditions.
  • the two main elements of the CyberKnife are (1) the radiation produced from a small linear particle accelerator and (2) a robotic arm which allows the energy to be directed at any part of the body from any direction.
  • the CyberKnife system is a method of delivering radiotherapy, with the intention of targeting treatment more accurately than standard radiotherapy.
  • High- Intensity Focused Ultrasound (HIFU) or "high frequency ultrasound” is a highly precise medical procedure using high- intensity focused ultrasound to heat and destroy pathogenic tissue rapidly by raising the tissue's immediate temperature in excess of 45°C. It is one modality of therapeutic ultrasound.
  • a "biopsy” is a medical test involving the removal of cells or tissues for examination. It is the medical removal of tissue from a living subject to determine the presence or extent of a disease. The tissue is generally examined under a microscope by a pathologist, and can also be analyzed chemically. When an entire lump or suspicious area is removed, the procedure is called an excisional biopsy. When only a sample of tissue is removed with preservation of the histological architecture of the tissue's cells, the procedure is called an incisional biopsy or core biopsy. When a sample of tissue or fluid is removed with a needle in such a way that cells are removed without preserving the histological architecture of the tissue cells, the procedure is called a needle aspiration biopsy.
  • a "radical prostatectomy” is the surgical removal of the prostate gland in order to remove prostate cancer.
  • Radiotherapy sometimes abbreviated to XRT, is the medical use of ionizing radiation, generally as part of cancer treatment to control malignant cells. Radiation therapy is commonly applied to the cancerous tumor because of its ability to control cell growth. Ionizing radiation works by damaging the DNA of exposed tissue, furthermore, it is believed that cancerous cells may be more susceptible to death by this process as many have turned off their DNA repair machinery during the process of becoming cancerous. To spare normal tissues (such as skin or organs which radiation must pass through in order to treat the tumor), shaped radiation beams are aimed from several angles of exposure to intersect at the tumor, providing a much larger absorbed dose there than in the surrounding, healthy tissue.
  • the radiation fields may also include the draining lymph nodes if they are clinically or radiologically involved with tumor, or if there is thought to be a risk of subclinical malignant spread. It is necessary to include a margin of normal tissue around the tumor to allow for uncertainties in daily set-up and internal tumor motion. These uncertainties can be caused by internal movement (for example, respiration and bladder filling) and movement of external skin marks relative to the tumor position.
  • clonotype is a collection of samples that are substantially similar and/or
  • FFPE Form-fixed, paraffin-embedded
  • an "adjuvant” is a pharmacological or immunological agent that modifies the effect of other agents, such as a drug or vaccine. They are often included in vaccines to enhance the recipient's immune response to a supplied antigen, while keeping the injected foreign material to a minimum. Adjuvants in immunology are often used to modify or augment the effects of a vaccine by stimulating the immune system to respond to the vaccine more vigorously, and thus providing increased immunity to a particular disease.
  • Adjuvants accomplish this task by mimicking specific sets of evolutionarily conserved molecules, so called PAMPs, which include liposomes, lipopolysaccharide (LPS), molecular cages for antigen, components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and unmethylated CpG dinucleotide-containing DNA.
  • dsRNA double-stranded RNA
  • ssDNA single-stranded DNA
  • CpG dinucleotide-containing DNA unmethylated CpG dinucleotide-containing DNA.
  • This example illustrates a protocol for collecting outcome information for different medical treatments of prostatic cancer patients, where each treatment includes use of autologous engineered T cells.
  • Patient eligibility criteria are as follows: histologically confirmed adenocarcinoma of the prostate. We obtain a clinically diverse set of patients in order to analyze the relationship between the appearance of induced clonotypes to the clinical characteristics of the patient. This diversity includes patients with localized and non- localized disease. This diversity includes patients with or without prior history of treatment (e.g., hormone deprivation treatment). This diversity includes a range of stages of the disease.
  • Medical procedures include the following: cryosurgery, radical prostatectomy, prostate biopsy, radiation therapy, brachytherapy, robotic-mediated radiotherapy using, for example, the CyberKnife Robotic Radiosurgery System (Accuray Incorporated, Sunnyvale, CA), electroporation, high frequency ultrasound (HIFU), photodynamic therapy, prostate laser surgery, androgen deprivation therapy, chemotherapy, and others.
  • prostate biopsies are typically considered to be diagnostic procedures rather than therapeutic procedures, but it is known that circulating tumor cells may increase in the days after a biopsy, so in this context we include it in our list of medical procedures of interest.
  • radical prostatectomies induce an immune response, even with the removal of tumor tissue, due to tumor tissue shedding caused by the surgery.
  • Sample collection is as follows: each sample consists of 10 niL of blood. We isolate peripheral blood mononuclear cells by by a standard centrifugation technique for cell separation on Ficoll-Hypaque Density Media (Sigma- Aldrich, St. Louis, MO).
  • FACS Fluorescence- Activated Cell Sorting
  • B cells CD8+ T cells, CD4+ T cells, CD4 Thl cells, CD4 Th2 cells, CD4 Thl7 cells, Treg cells (nTreg, iTreg, Th3, Trl), NKT cells, and/or gamma-delta T cells.
  • CD8+ T cells CD4+ T cells
  • CD4 Thl cells CD4 Th2 cells
  • CD4 Thl7 cells CD4 Thl7 cells
  • Treg cells nTreg, iTreg, Th3, Trl
  • NKT cells gamma-delta T cells.
  • gamma-delta T cells gamma-delta T cells
  • induced clonotypes From sequence data of the complementarily-determining region 3 (CDR3) of the T cell receptor (TCR) provided by the sequencing vendor, we identify induced clonotypes, as follows.
  • CDR3 complementarily-determining region 3
  • TCR T cell receptor
  • clonotypes We rank and characterize clonotypes as weakly or strongly emergent based on their percentage increase in frequency. For example, a clonotype which had a frequency of 0.1% prior to intervention and 0.9% after intervention has an increase in frequency of 0.8%; since this is greater than the minimum increase of 0.5%, we characterize this clonotype as induced.
  • lymphocyte receptors With induced or expanded clonotypes, we further characterize the lymphocyte receptors by the following criteria: Several receptors consist of two chains, which are paired in vivo. For example, in T cells, a receptor may consist of an alpha and a beta chain; a different receptor may consist of a gamma and a delta chain. In B cells, the two chains are the heavy chain and the light chain. In the following explanation, for convenience, we refer to alpha and beta chains, but a similar strategy is used for pairing heavy chains and light chains, or gamma chains and delta chains.
  • V and J gene segment-specific antibodies are readily available from commercial sources, including Life Technologies Corporation (Grand Island, NY). Therefore, we start with one chain - for example, the beta chain.
  • IMGT International IMmunoGeneTics information system
  • Lefranc Lefranc, et al., NUCL. ACIDS RES. 37:D 1006- D1012 (2009)
  • V-Quest Brochet, et al., NUCL. ACIDS RES. 36: W503-508 (2008)
  • JunctionAnalysis Yousfi Monod, et al., BlOINFORMATICS 20:i379-i385 (2004)
  • JOINSOLVER Souto- Carneiro, et al., J. IMMUNOL. 172(1 1):6790-6802 (2004)
  • VDJSolver Ohm-Laursen, et al.,
  • each microwell has a very low number of cells of between about 10 and 1.
  • an autologous B or T cell which expresses a receptor corresponding to the induced clonotype.
  • This receptor may be a T cell receptor on a T cell surface, or a chimeric antibody receptor.
  • the chimeric antibody receptor is comprised of a single chain variable fragment (scFv) on the T cell surface.
  • the chimeric antibody is enhanced by the presence of co-stimulatory endodomains.
  • vectors can be usefully employed including, for example, a gamma retroviral vector in view of mounting data showing these vectors to be safe when expressed in human T cells, for example. See Powell and Levine, "Genetically Engineered Antigen Specificity in T Cells for Adoptive Immunotherapy," in J. Medin and D. Fowler, Eds., Experimental and Applied Immunotherapy, Chapter 12 (Humana Press, 201 1).
  • Another alternative for engineering the autologous T cells is to introduce the foreign DNA specific to the TCR of interest using electroporation-mediated DNA or mRNA
  • transfection which are methodologies that are well-known to the skilled artisan. Recent reports have indicated, for example, greater than 90% transduction efficiency introducing tumor antigen-specific TCR genes that conferred tumor reactivity to previously nonreactive, unstimulated human T cells. Id.
  • cytokines such as IL-2 or IL-7 are used to facilitate this process; in this case, we follow vendor instructions.
  • T cells we test our engineered T cells in vitro by incubating them with tumor tissue lysate and also with various combinations of adjuvants such as GM-CSF, IL-2, and others.
  • adjuvants such as GM-CSF, IL-2, and others.
  • cytokines e.g., IFN-gamma
  • T cell proliferation we measure efficacy using assays for cytokines (e.g., IFN-gamma) and T cell proliferation. These assays are commercially available, and we follow vendor instructions.
  • Dunning R3327 rat prostatic tumor tissue
  • the Dunning R3327 rat carcinoma is a well-recognized model for human prostate adenocarcinoma. Sinowatz et al., PROSTATE 19(4):273-278 (1991).
  • the rats used for the procedure were Male Copenhagen rats between 8 and 15 weeks of age.
  • the rats and rat food were purchased from Harlan Teklad (Madison, WI).
  • the Dunning Cell line was obtained from Dr. Israel Barken (San Diego, CA) and propagated in RPMI 1640 media (Sigma-Aldrich, St. Louis, MO) with 10% Fetal Bovine Serum (FBS; Sigma-Aldrich, St. Louis, MO) in 5% C0 2 in air at 37°C.
  • Tumor cells were subcutaneously implanted in rats with 2 xlO 5 cells on the left side of the rat (about 1 ⁇ 2 inch from the inguinal area) and 2 x 10 4 cells on the right side. When tumors on the right side reached about 5 mm 3 to 7 mm 3 in size the rats were divided into four groups. Table 1 depicts the number and purpose of each group.
  • each rat from groups 3 and 4 were injected subcutaneously with 5000 injection units (IU) of interleukin-2 (IL-2) and 1000 IU of granular macrophage colony stimulating factor (GM-CSF) approximately 1 ⁇ 4 inch from the tumor sit towards the inguinal node on the right side.
  • IL-2 interleukin-2
  • GM-CSF granular macrophage colony stimulating factor
  • Tumor dimensions were measured 3 times a week for 60 days by measuring the perpendicular minor dimension (W) and major dimension (L) using sliding calipers. Approximate tumor volume was calculated by the formula W 2 x L x 1 ⁇ 2.
  • Table 3 illustrates that cryosurgery was more effective against the Dunning 3327 prostate cancer when combined with the immunotherapeutics IL-2 and GM-CSF.
  • Table 4 shows a tumor cure rate in the rats receiving the combined treatment reached 80%.
  • Table 5 shows that the rats receiving the combined treatment had a significant increase in survival time as compared to the rats in other groups.
  • IL-2 IL-2 and GM-CSF. This model may prove to be effective for human prostate adenocarcinomas.
  • cryosurgical procedures may include other procedures such as radical prostatectomy, prostate biopsy, radiation therapy, brachytherapy, robotic-mediated radiosurgery using, for example, the CyberKnife Robotic Radiosurgery System (Accuray Incorporated, Sunnyvale, CA), electroporation, high frequency ultrasound (HIFU), photodynamic therapy, prostate laser surgery, androgen deprivation therapy, chemotherapy, and others.
  • the range of cytokines used is also not to be understood as limited to IL-2 and GM-CSF and may include IL-4, IL-6, IFN- ⁇ , and others.
  • This example illustrates a protocol for identifying lymphocyte clonotypes that are usefully employed in one embodiment of the present invention.
  • Heparinized venous blood is layered onto a Ficoll-Isopaque (Lympho separation Medium, ICN Bio medicals, Ohio) in a 15 mL conical tube in the ration of 3 : 1.
  • the tubes are centrifuged at 1800 rpm for 20 minutes and the middle layer is removed and transferred to another tube.
  • the middle layer is then centrifuged at 2000 rpm for 10 minutes.
  • the supernatant is discarded and the pellet is suspended in RPMI-1640 provided by ICN Bio chemicals, Ohio.
  • the cells are washed twice with RPMI-1640 and suspended in lmL of RPMI-1640.
  • the cell suspensions are divided into two tubes and incubated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labeled anti-CD4 or anti-CD8 monoclonal antibodies manufactured by Pharmingen, San Jose, CA.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • the tubes are mildly vortexed and incubated for 2 hours in the dark at room temperature.
  • CD4 + and CD8 + cells are identified based on staining with specific antibodies and sorted on Fluorescence- Activated Cell Sorting (FACS) Star Plus manufactured by Becton Dickinson, San Jose, CA.
  • FACS Fluorescence- Activated Cell Sorting
  • This example illustrates basis for identifying B cell or T cell mixtures that include those having receptors that have increased in abundance using non- sequencing methods.
  • the general strategy of the present invention is to study the immune system's response to an event that triggers an immune response, necessarily starting with what is referred to herein as an immune recognition event.
  • Immune recognition events can be induced by injury or a medical procedure or intervention or other event. Studying the result of the immune recognition event provides the information needed to design and produce a tailored therapeutic intervention that, in one embodiment, induces a stronger and more directed immune response to the antigens that the immune system itself organized itself to attack.
  • One embodiment of the present invention is to sequence the T and/or B cell receptor repertoire and then genetically engineer a T cell- and/or B cell-based therapy for infusion into the patient whose receptor repertoire was studied.
  • Another embodiment of this approach replaces T cell receptor or B cell receptor sequencing with spectratyping (also known as immunoscoping).
  • Spectratyping analysis is a method for quantifying the presence of lymphocytes with particular V and/or J segments, as set forth in Kepler et al., "Statistical Analysis of Antigen Receptor Spectratype Data," BIOINFORMATICS 21(16):3394-3400 (2005) at Fig. 1 on p. 3395.
  • spectratyping analysis There are multiple ways to carry out spectratyping analysis, including quantitative PCR, flow cytometry with V and/or J segment specific antibodies, and the like. It is important to note that some forms of spectratyping reveal not just the quantity of cells with a given V and J segment, but also the oligoclonality within each particular combination of V and/or J segments.
  • the quantification of V and/or J segments can also be done using sequencing - starting with the sequence reads, the V and J segments can be calculated in silico (e.g. using IMGT tools such as V-Quest and IMGT HighV-Quest, which were described and referenced above).
  • One advantage of spectratyping is that it is faster than sequencing.
  • One advantage of sequencing is that it reveals the diversity (i.e., the oligoclonality) of an individual V-J "compartment.”
  • a given subpopulation of lymphocytes expressing a particular V and/or a particular J segment may be composed of mostly one expanded clone or a large number of different cells from different clones.
  • Sequencing data clarifies the content of the V-J compartment - but a perfectly valid and useful
  • immunotherapeutic cell-based reagent can be identified and used without necessarily having the sequence data.
  • Another embodiment of the present invention replaces the genetic engineering step with a filtering / purification step, in which the patient's own lymphocytes are filtered to isolate lymphocytes bearing a particular V and/or J segment.
  • This filtering can be done readily using a variety of technologies well-known in the art, including FACS, magnetic beads, and others. These techniques are enabled by commercially- available antibodies to various human V and J segments from, for example, Pierce Antibodies from Thermo Fisher Scientific (Rockford, IL).
  • a first objective is to identify those T cells or B cells, or both, that increase in abundance in a patient's blood after an immune recognition event, such as a medical procedure, intervention or event; and then identify a particular combination of V and/or J segments in the T cell or B cell repertoires that has expanded and/or become more oligoclonal.
  • a subset of cells expressing the identified V and/or J segments is then isolated from the patient's own blood, expanded, stimulated, or modified ex vivo, and then reinfused as a therapeutic treatment.
  • B cell repertoires which has expanded and/or become more oligoclonal is identified.
  • a subset of cells expressing the identified V and/or J segments is then isolated from the patient's own blood, expanded, stimulated, and then reinfused as a therapeutic treatment.
  • the protocol using spectratyping to identify one or more lymphocyte groupings that expanded in abundance after the immune recognition response has an optional step for modifying the autologous lymphocyte ex vivo.
  • Modifications usefully employed in the context of the present invention include engineering the autologous lymphocytes to tweak the TCR or BCR activity by adding a CAR, for example.
  • a key advantage of this protocol using spectratyping to identify the cell-based immune-reagent composed of a particular V and/or J subset of lymphocytes, is that the isoation step can be done very quickly (relativeto sequence based approaches requiring engineering of the autologous lymphocytes - not as an option but a necessary step).
  • Another variation on this theme is to identify an expanded receptor using before and after immune recognition event sequencing of DNA from the sets of lymphocytes, then carry out a "panning experiment" in which many copies of the identified receptor are incubated with a combinatorial library of possible epitopes.
  • This approach relies on methods well-known in the art, including Sung et al., "T-Cell Epitope Prediction with Combinatorial Peptide Libraries,” J. COMPUT. BIOL. 90 ⁇ :527-39 (2002); and Pira et al., "High Throughput T Epitope Mapping and Vaccine Development," J. BlOMED. BlOTECHNOL. 2010:325720 (2010).
  • lymphocytes from the patient's own cells which are specific to that epitope (which should be mainly composed of the expanded receptor identified previously). These cells are then expanded ex vivo, manipulated (e.g. with cytokines), and then therapeutically infused into the patient.

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Abstract

Un exemple de la présente invention porte sur une agression délibérée ou sur la reconnaissance qu'une agression s'est produite chez un patient, ladite agression induisant un événement de reconnaissance par le système immunitaire. Le répertoire des lymphocytes T est catalogué à partir d'échantillons des lymphocytes du patient avant et après l'agression afin de détecter et de séquencer les loci TCR alpha et bêta de clonotypes de lymphocytes T fortement développés. Dans certains exemples, cette information est utilisée à son tour pour générer des lymphocytes T autologues ou pour créer des lymphocytes T génétiquement modifiés autologues, les deux types comprenant des séquences TCR qui ciblent la maladie ou la lésion de l'individu, et soignent ou améliorent ainsi les symptômes.
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