Ex vivo methods for generating T cell therapeutics and related compositions and methods

Abstract

Methods for the ex vivo expansion of T cells, including tumor-reactive T cells, and compositions containing such T cells are provided herein. Methods for treating diseases and conditions, such as cancer, using the compositions of the present disclosure are also provided.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 826,974, filed March 29, 2019, entitled "EX VIVO METHODS FOR PRODUCING AT CELL THERAPEUTIC AND RELATED COMPOSITIONS AND METHODS," U.S. Provisional Patent Application No. 62 / 941,610, filed November 27, 2019, entitled "EX VIVO METHODS FOR PRODUCING AT CELL THERAPEUTIC AND RELATED COMPOSITIONS AND METHODS," and U.S. Provisional Patent Application No. 62 / 978,298, filed February 18, 2020, entitled "EX VIVO METHODS FOR PRODUCING AT CELL THERAPEUTIC AND RELATED COMPOSITIONS AND METHODS," the contents of each of which are incorporated by reference in their entirety.

[0002] Incorporation by reference of sequence listing This application is filed with an electronic Sequence Listing. The Sequence Listing is provided as a file entitled 165172000240SeqList.txt, created on March 26, 2020, and is 5 kilobytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure provides methods for the ex vivo expansion of T cells, including tumor-reactive T cells, and compositions containing such T cells. Methods for treating diseases and conditions, such as cancer, using the compositions of the present disclosure are also provided. [Background technology]

[0004] background Cancer cells accumulate many different DNA mutations as part of the tumorigenesis process. These mutations can cause amino acid changes in protein-coding regions. For the mutations to be recognized by the immune system, the protein must be processed intracellularly and present a mutant peptide on the surface via the major histocompatibility complex (MHC). Neoantigens are mutant peptides presented by the MHC complex that can be recognized by T cells via TCR binding. Neoantigens are ideal targets for immunotherapy. These antigens are not present in the body before cancer develops, are truly cancer-specific, are not expressed on normal cells, and are not subject to off-target immunotoxicity. Clinical trials have demonstrated that T cells isolated from surgically resected tumors possess TCRs that recognize neoantigens, and expanding these neoantigen-reactive TIL populations and reinfusing them into patients can sometimes result in dramatic clinical benefits. However, in cell therapy, a major obstacle to the application of such cells is the difficulty of obtaining such cells. Improved methods for obtaining and manufacturing cell compositions containing tumor-reactive T cells for therapeutic use are needed. Embodiments that meet this need are provided herein. Summary of the Invention

[0005] overview Provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) incubating a cell population containing T cells derived from a biological sample obtained from a subject with a T cell stimulator under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens obtained from the subject, thereby generating a T cell-containing population containing tumor-reactive T cells; (c) enriching the co-culture for a tumor-reactive T cell population reactive to the one or more peptides; and (d) incubating the tumor-reactive T cell-enriched T cell population with a T cell stimulator under conditions to stimulate expansion of T cells within the population, wherein one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor. In some embodiments, the methods further comprise (e) harvesting the cells generated by the method.

[0006] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) (a) incubating a cell population comprising T cells from a biological sample obtained from a subject bearing a tumor with a first T cell stimulator under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens obtained from the subject, thereby generating a T cell-containing population comprising tumor-reactive T cells; and (d) enriching the co-culture for tumor-reactive T cells that are reactive to the one or more peptides, thereby enriching for tumor-reactive T cells. (e) culturing the T cells by a process comprising incubating the tumor-reactive T cell enriched T cell population with a second T cell stimulator under conditions to stimulate expansion of T cells within the population, wherein one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor that inhibits caspase activation or activity; and (2) harvesting the cells produced by the method to produce a composition of expanded T cells enriched for tumor-reactive T cells.

[0007] In some of any of the provided embodiments, the first or second T cell stimulator comprises the presence of one or more recombinant cytokines, and the incubation in the presence of the T cell stimulator occurs before, during, and / or after incubation in the presence of one or more T cell adjuvants.

[0008] In some of any of the provided embodiments, the incubation with at least one T cell adjuvant occurs during at least a portion of the co-culture. In some of any of the embodiments, at least a portion of the incubating of the T cell population with the at least one T cell adjuvant occurs simultaneously with incubating the T cell population with the T cell stimulator.

[0009] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) (a) incubating a cell population comprising T cells derived from a biological sample from a subject bearing a tumor with a first T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant that is an apoptosis inhibitor that inhibits caspase activation or activity; (b) incubating a cell population comprising T cells derived from a tumor-associated antigen from the subject in the presence of antigen-presenting cells (APCs); or a plurality of peptides, thereby producing a population containing T cells that includes tumor-reactive T cells; (c) enriching the co-culture for tumor-reactive T cells that contain endogenous TCRs reactive to tumor-associated antigens, thereby producing a T cell population enriched for tumor-reactive T cells; (d) culturing the T cells by a process comprising incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of cells of the cell population; and (2) harvesting the cells produced by the method.

[0010] In some of the provided embodiments, the at least one T cell adjuvant further comprises at least one costimulatory agonist. In some of the provided embodiments, the incubating with the at least one costimulatory agonist and the apoptosis inhibitor is performed simultaneously, intermittently, or sequentially.

[0011] In some of any of the provided embodiments, the at least one T cell adjuvant further comprises at least one checkpoint inhibitor. In some of any of the provided embodiments, the incubating with the at least one checkpoint inhibitor and the apoptosis inhibitor occurs simultaneously, intermittently, or sequentially.

[0012] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) (a) incubating a cell population comprising T cells derived from a biological sample obtained from a subject bearing a tumor with a first T cell stimulator under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens obtained from the subject, thereby generating a T cell-containing population comprising tumor-reactive T cells; and (d) enriching the tumor-reactive T cell population reactive to the one or more peptides from the co-culture, and (d) culturing the T cells by a process comprising incubating the tumor-reactive T cell enriched T cell population with a second T cell stimulator under conditions to stimulate expansion of T cells within the population, wherein one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptotic agent; and (b) harvesting the cells produced by the method to produce a composition of expanded T cells enriched for tumor-reactive T cells.

[0013] In some of any of the provided embodiments, the first or second T cell stimulator comprises the presence of one or more recombinant cytokines, and the incubation in the presence of the T cell stimulator occurs before, during, and / or after incubation in the presence of one or more T cell adjuvants.

[0014] Provided herein are methods for producing tumor-reactive T cells, the methods comprising: (1) culturing the T cells by a process comprising incubating an ex vivo population of T cells, comprising tumor-reactive T cells comprising an endogenous TCR reactive to a tumor-associated antigen, in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, wherein the T cell population is enriched for CD4+ T cells and CD8+ T cells, and wherein at least a portion of the incubation occurs before, simultaneously with, or after incubating the T cells with a T cell stimulatory agent under conditions to activate the T cells, and wherein the culturing stimulates expansion of cells of the T cell population; and (2) harvesting the cells produced by the method.

[0015] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) (a) incubating a cell population comprising T cells from a biological sample from a subject having a tumor with a first T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant that is a costimulatory agonist; (b) incubating a cell population comprising T cells from a biological sample from a subject having a tumor with a first T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population; (c) co-culturing the stimulated T cell population under conditions in which APCs are induced to express, thereby generating a population containing T cells, including tumor-reactive T cells; (c) enriching the co-culture for tumor-reactive T cells that contain endogenous TCRs reactive to the tumor-associated antigen, thereby generating a T cell population enriched for tumor-reactive T cells; (d) culturing the T cells by a process comprising incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of cells of the cell population; and (2) harvesting the expanded cells generated by the method.

[0016] In some of any of the provided embodiments, the at least one T cell adjuvant further comprises at least one apoptosis inhibitor. In some of any of the provided embodiments, the at least one apoptosis inhibitor and the checkpoint agonist are administered simultaneously, intermittently, or sequentially. In some of the provided embodiments, the at least one T cell adjuvant further comprises at least one checkpoint inhibitor. In some of the embodiments, the at least one checkpoint inhibitor and the costimulatory agonist are administered simultaneously, intermittently, or sequentially.

[0017] Provided herein are methods for producing tumor-reactive T cells, the methods comprising: (1) culturing the T cells by a process comprising: (a) incubating an ex vivo population of T cells, the ex vivo population of T cells comprising tumor-reactive T cells comprising an endogenous TCR reactive to a tumor-associated antigen, with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, wherein the T cell population is enriched for CD4+ T cells and CD8+ T cells; and (b) incubating the T cells with a T cell stimulator under conditions to activate the T cells, wherein at least a portion of the incubating occurs before, simultaneously with, or after the incubating in (a), wherein the culturing stimulates expansion of cells of the T cell population; and (2) harvesting the cells produced by the method.

[0018] Provided herein is a method for producing tumor-reactive T cells, the method including: (1) (a) incubating a cell population comprising T cells from a biological sample obtained from a subject having a tumor with a first T cell stimulator under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens obtained from the subject, thereby generating a population containing T cells comprising tumor-reactive T cells; and (d) extracting from the co-culture T cells reactive to the one or more peptides. (d) enriching a tumor-reactive T cell population that is enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of T cells within the population, wherein one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant that is a checkpoint inhibitor; and (e) harvesting the expanded cells produced by the method.

[0019] In any of the embodiments, the step of culturing the T cells further comprises incubating the cells in the presence of one or more recombinant cytokines, where the incubation in the presence of the one or more cytokines occurs before, during, and / or after the incubation with the T cell stimulant and / or the incubation in the presence of one or more T cell adjuvants.

[0020] In some of any of the provided embodiments, the first or second T cell stimulator comprises the presence of one or more recombinant cytokines, and the incubation in the presence of the T cell stimulator occurs before, during, and / or after the incubation in the presence of one or more T cell adjuvants. In some of the provided embodiments, the incubation with at least one T cell adjuvant occurs during at least a portion of the co-culture. In some of the provided embodiments, at least a portion of the incubating of the T cell population with the at least one T cell adjuvant occurs simultaneously with the incubating of the T cell population with the T cell stimulator.

[0021] In some of any of the embodiments, the T cell stimulatory agent comprises the presence of one or more recombinant cytokines, and the incubation in the presence of the T cell stimulatory agent occurs before, during, and / or after incubation in the presence of one or more T cell adjuvants.

[0022] In some of any of the embodiments, the T cell agent comprises an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling through a costimulatory receptor, optionally the costimulatory receptor is CD28, and the incubating in the presence of the T cell agent occurs before, during, and / or after the incubating in the presence of one or more T cell adjuvants.

[0023] In some of any of the provided embodiments, the methods further include washing the cells during one or more of the culturing steps, e.g., during incubation with a T cell stimulator and / or one or more T cell adjuvants, e.g., a costimulatory agonist, an apoptosis inhibitor (e.g., a caspase inhibitor), or a checkpoint inhibitor. In some embodiments, the provided methods include adding a transient addition (e.g., only once, such as at the beginning of incubation or culturing) of a T cell adjuvant, e.g., a costimulatory agonist, an apoptosis inhibitor (e.g., a caspase inhibitor), or a checkpoint inhibitor, during one or more of the steps. In some embodiments, the provided methods include continuous addition of a T cell adjuvant during one or more of the steps. For example, one or more T cell adjuvants, e.g., a costimulatory agonist, an apoptosis inhibitor (e.g., a caspase inhibitor), or a checkpoint inhibitor, are added continuously during one or more of the steps of incubating the cells, e.g., during incubation with a T cell stimulator. For example, during or after one or more steps, cell is washed, and then culture medium is supplemented with T cell adjuvant at the same concentration.In some cases, one or more T cell stimulants are also supplemented together with washing.During this step, for example, during incubation with one or more T cell stimulants, washing can be carried out every day, every other day, every two days, every three days, every four days, every five days, or once a week.

[0024] In some of any of the embodiments, the method further comprises washing the cells after one or more of the steps of incubating the cells with a T cell stimulator, at least one T cell adjuvant, and / or one or more recombinant cytokines.

[0025] In some of any of the embodiments, the culturing step is carried out until a threshold amount of cells is obtained and / or for up to 20 days.

[0026] In some of any of the embodiments, the T cell population comprising tumor-reactive T cells comprises T cells that are surface positive for one or more T cell activation markers selected from the group consisting of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3. In some of any of the embodiments, the T cell population comprising tumor-reactive T cells is enriched for T cells that are surface positive for one or more T cell activation markers selected from the group consisting of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3. In some of any of the provided embodiments, the T cell population comprising tumor-reactive T cells is enriched for tumor-reactive T cells.

[0027] In some of any of the provided embodiments, the T cells comprise primary T cells from a subject, optionally a human subject.

[0028] In some of any of the provided embodiments, the T cell population comprising tumor-reactive T cells is present in an ex vivo co-culture comprising autologous T cells derived from a biological sample from the subject and antigen-presenting cells (APCs), wherein the co-culture is performed under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject. In some of any of the provided embodiments, a T cell population comprising tumor-reactive T cells is generated by a process comprising: (a) identifying somatic mutations associated with one or more tumor-associated antigens by exome sequencing of healthy and tumor tissue from the subject; (b) identifying at least one epitope of the one or more tumor-associated antigens, where optionally, the at least one epitope is a neoepitope; (c) isolating an autologous T cell population from a biological sample from the subject; and (d) co-culturing the T cell population with antigen-presenting cells (APCs) that have been exposed to or contacted with one or more peptides comprising at least one epitope of the one or more tumor-associated antigens under conditions for presenting one or more of the peptides on the surface of a major histocompatibility complex (MHC), thereby generating a T cell population comprising tumor-reactive T cells that are reactive to the one or more peptides.

[0029] In some embodiments, exposing or contacting APCs with one or more peptides comprises generating a mutant library of peptides comprising one or more epitopes, optionally wherein the peptides are 8-32 amino acids in length, 8-24 amino acids in length, 8-18 amino acids in length, 8-10 amino acids in length, 10-32 amino acids in length, 10-24 amino acids in length, 10-18 amino acids in length, 18-32 amino acids in length, 18-24 amino acids in length, or 24-32 amino acids in length, and optionally are 9-mers or about 9-mers; and pulsing APCs with the mutant library of peptides under conditions to present one or more of the peptides on the surface of a major histocompatibility complex (MHC), optionally wherein the MHC is MHC class I. In some embodiments, exposing or contacting an APC with one or more peptides comprises generating DNA, optionally a minigene construct, encoding one or more tumor-associated antigens, or portions thereof, including somatic mutations; in vitro transcribing the DNA into RNA; and introducing the in vitro transcribed RNA into the APC under conditions for presenting one or more of the peptides on the surface of a major histocompatibility complex (MHC), optionally where the MHC is MHC class II.

[0030] In some embodiments, the co-culture is carried out for 1 to 7 days. In some embodiments, incubation with at least one T cell adjuvant is carried out during at least a portion of the co-culture. In some embodiments, the method further comprises selecting T cells reactive to one or more peptides from the co-culture, thereby enriching the T cell population including tumor-reactive T cells. In some embodiments, the enriching step is carried out before incubation with at least one T cell adjuvant. In some embodiments, incubation with a T cell stimulator is carried out before co-culture, and an autologous T cell population derived from a biological sample from the subject is incubated with the T cell stimulator. In some embodiments, incubation with a T cell stimulator is carried out after enriching the T cell population including tumor-reactive T cells. In some embodiments, the APCs are autologous to the subject or allogeneic to the subject.

[0031] In some of the provided embodiments, the co-culture ratio of antigen-presenting cells to T cells is 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 5:1 to 1:1, 2.5:1 to 1:1, 1:20 to 1:1, 1:15 to 1:1, 1:10 to 1:1, 1:5 to 1:1, or 1:2.5 to 1:1. In some of the provided embodiments, the co-culture ratio of antigen-presenting cells to T cells is 1:1 or about 1:1. In some of the provided embodiments, the co-culture is for 2 to 24 hours. In some of the provided embodiments, the co-culture is for 6 hours or about 6 hours.

[0032] In some of any of the provided embodiments, the subject is healthy or not known to exhibit a disease or condition.

[0033] In some of any of the provided embodiments, the culturing step includes isolating a cell population comprising T cells from a biological sample from the subject; incubating the cell population comprising T cells with a T cell stimulant under conditions that activate T cells of the population to produce a T cell population comprising activated cells; co-culturing the T cell population comprising activated cells in the presence of APCs under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby producing a T cell population comprising tumor-reactive T cells, wherein the co-culturing is performed in the presence of at least one T cell adjuvant, thereby producing a T cell population comprising tumor-reactive T cells with the at least one adjuvant. selecting from the co-culture T cells that are reactive to one or more peptides, thereby enriching the T cell population containing tumor-reactive T cells; and further incubating the cells in the presence of one or more recombinant cytokines, optionally before, during, and / or after incubating with a T cell stimulator, co-culturing in the presence of APCs, and / or incubating in the presence of one or more T cell adjuvants, wherein the culturing step stimulates expansion of cells of the T cell population.

[0034] In some of any of the provided embodiments, the culturing step comprises isolating a cell population comprising T cells from a biological sample from the subject; incubating the cell population comprising T cells with a T cell stimulant under conditions that activate T cells of the population to produce a T cell population comprising activated cells; co-culturing the T cell population comprising activated cells in the presence of APCs under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby producing a T cell population comprising tumor-reactive T cells; selecting T cells from the co-culture that are reactive to the one or more peptides, thereby enriching the T cell population comprising tumor-reactive T cells; incubating the enriched population of T cells comprising tumor-reactive cells in the presence of at least one T cell adjuvant; and further incubating the cells in the presence of one or more recombinant cytokines, optionally wherein the incubating in the presence of the one or more cytokines occurs before, during, and / or after the incubating with the T cell stimulant, the co-culturing in the presence of APCs, and / or the incubating in the presence of one or more T cell adjuvants, and wherein the culturing step stimulates expansion of cells of the T cell population.

[0035] In some of any of the provided embodiments, the culturing step includes isolating a cell population comprising T cells from a biological sample from the subject; co-culturing the T cell population in the presence of APCs under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a T cell population comprising tumor-reactive T cells; incubating the population comprising tumor-reactive T cells with a T cell stimulator under conditions that activate T cells of the population to generate a T cell population comprising activated cells; and selecting T cells from the co-culture that are reactive to the one or more peptides, thereby enriching the T cell population comprising tumor-reactive T cells, wherein selecting incubating the T cell population enriched for tumor-reactive T cells in the presence of at least one T cell adjuvant; and further incubating the cells in the presence of one or more recombinant cytokines, optionally wherein the incubation in the presence of one or more cytokines occurs before, during, and / or after the incubation with the T cell stimulant, co-culturing in the presence of APCs, and / or incubation in the presence of one or more T cell adjuvants, and the culturing step stimulates expansion of cells of the T cell population.

[0036] In some of any of the provided embodiments, the co-cultivation occurs for 1 to 7 days.

[0037] In some of the provided embodiments, the T cell population comprising tumor-reactive T cells is isolated or directly selected from a biological sample from a subject. In some of the provided embodiments, the subject exhibits a disease or condition, and optionally, the disease or condition is cancer. In some of the provided embodiments, the subject has previously been administered a costimulatory agonist prior to isolating or selecting the T cell population.

[0038] In some of any of the provided embodiments, the T cell population comprising tumor-reactive T cells is allogeneic to the subject being treated or autologous to the subject being treated, and optionally, the subject being treated has a tumor or cancer. In some of any of the provided embodiments, the biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample.

[0039] In some of the provided embodiments, the biological sample is a peripheral blood sample, and the peripheral blood sample is collected by blood draw or apheresis, and optionally, the apheresis is leukapheresis. In some of the provided embodiments, the biological sample has a volume of 50 mL to 400 mL. In some of the provided embodiments, enriching for tumor-reactive T cells or T cells surface-positive for one or more activation markers is by flow cytometry, optionally by automated high-throughput flow cytometry. In some embodiments, enriching can be performed using an FX500 cell sorter. In some of the provided embodiments, one, two, three, or four runs of flow cytometry are performed to enrich for tumor-reactive T cells from the sample.

[0040] In some of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, and the sample is collected by needle biopsy, optionally by core needle biopsy or fine needle aspiration. In some of the provided embodiments, the biological sample comprises tumor fragments. In some of the provided embodiments, the T cell population comprises tumor-infiltrating lymphocytes.

[0041] Provided herein are methods for producing tumor-reactive T cells, the method comprising: (1) culturing the T cells by a process comprising: (a) enriching tumor-reactive T cells comprising an endogenous TCR reactive to a tumor-associated antigen from a biological sample containing T cells, thereby generating a T cell population enriched in tumor-reactive T cells; and (b) incubating the T cell population enriched in tumor-reactive T cells in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, wherein at least a portion of the incubation occurs before, simultaneously with, or after incubating the T cell population with a T cell stimulator under conditions to stimulate expansion of cells of the T cell population, wherein the culturing stimulates expansion of cells of the T cell population; and (2) harvesting the cells produced by the method.

[0042] In some aspects, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (1) culturing the T cells by a process comprising: (a) enriching tumor-reactive T cells comprising an endogenous TCR reactive to a tumor-associated antigen from a biological sample comprising T cells, thereby generating a T cell population enriched in tumor-reactive T cells; (b) incubating the T cell population enriched in tumor-reactive T cells with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; and (c) incubating the T cell population enriched in tumor-reactive T cells with a T cell stimulator under conditions to stimulate expansion of cells of the cell population, wherein at least a portion of the incubating occurs before, simultaneously with, or after the incubating in (b), wherein the culturing stimulates expansion of cells of the T cell population; and (2) harvesting the cells produced by the method.

[0043] In some of any of the provided embodiments, culturing the T cells further comprises incubating the cells in the presence of one or more recombinant cytokines, where the incubating in the presence of the one or more cytokines occurs before, during, and / or after the incubating with the T cell stimulant and / or the incubating in the presence of one or more T cell adjuvants.

[0044] In some of any of the embodiments, the T cell stimulator comprises the presence of one or more recombinant cytokines, and the incubation in the presence of the T cell stimulator occurs before, during, and / or after incubation in the presence of one or more T cell adjuvants.

[0045] In some of any of the embodiments, the T cell agent comprises an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling through a costimulatory receptor, optionally the costimulatory receptor is CD28, and the incubating in the presence of the T cell agent occurs before, during, and / or after the incubating in the presence of one or more T cell adjuvants.

[0046] In some of any of the provided embodiments, the method further comprises washing the cells after one or more of the steps of incubating the cells with the T cell stimulator, at least one T cell adjuvant, and / or one or more recombinant cytokines. In some of any of the provided embodiments, the culturing is carried out until a threshold amount of cells is obtained and / or for up to 20 days.

[0047] In some of any of the provided embodiments, at least a portion of the incubating of the T cell population with the at least one T cell adjuvant is performed simultaneously with incubating the T cell population with the T cell stimulator.

[0048] In some of any of the provided embodiments, incubating the T cell population with at least one T cell adjuvant occurs after incubating the T cell population with a T cell stimulator.

[0049] In some of any of the provided embodiments, the culturing step includes enriching the biological sample containing T cells for tumor-reactive T cells that contain endogenous TCRs reactive to a tumor-associated antigen, thereby generating a T cell population enriched for tumor-reactive T cells; incubating the T cell population containing the reactive T cells with a T cell stimulator under conditions that activate the T cells of the population to generate a T cell population containing activated cells; incubating the T cell population containing the activated T cells in the presence of at least one T cell adjuvant; and further incubating the cells in the presence of one or more recombinant cytokines, optionally wherein the incubating in the presence of the one or more cytokines occurs before, during, and / or after the incubating with the T cell stimulator and / or the incubating in the presence of one or more T cell adjuvants, and the culturing step stimulates expansion of cells of the T cell population. In some of any of the provided embodiments, the incubating in the presence of at least one T cell adjuvant occurs 1 to 5 days before the step of harvesting the cells.

[0050] In some of any of the provided embodiments, incubating the T cell population with at least one T cell adjuvant occurs before incubating the T cell population with a T cell stimulator. In some of the provided embodiments, enriching for tumor-reactive T cells and / or T cells expressing one or more T cell activation markers occurs after incubating the T cell population with at least one T cell adjuvant and before incubating the T cell population with a T cell stimulator, wherein the enriched T cell population is incubated with the T cell stimulator.

[0051] In some of any of the provided embodiments, the T cells are primary T cells from a subject, optionally a human subject.

[0052] In some of the provided embodiments, the biological sample is an ex vivo co-culture comprising autologous T cells from the subject and antigen-presenting cells (APCs), wherein the co-culture is performed under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens from the subject. In some of the provided embodiments, the APCs are autologous to the subject or allogeneic to the subject. In some of the provided embodiments, the subject is healthy or not known to exhibit a disease or condition.

[0053] In some of the provided embodiments, the biological sample is obtained from a subject, and selecting includes directly isolating T cells from the biological sample. In some of the provided embodiments, the subject exhibits a disease or condition, and optionally, the disease or condition is cancer. In some of the provided embodiments, the subject has previously been administered a costimulatory agonist prior to isolating or selecting the T cell population. In some of the provided embodiments, the T cell population is allogeneic or autologous to the subject being treated, and optionally, the subject being treated has a tumor or cancer. In some of the provided embodiments, the biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample.

[0054] In some of the provided embodiments, the biological sample is a peripheral blood sample, and the peripheral blood sample is collected by blood draw or apheresis, and optionally, the apheresis is leukapheresis. In some of the provided embodiments, the biological sample has a volume of 50 mL to 400 mL. In some of the provided embodiments, enriching for tumor-reactive T cells or T cells surface-positive for one or more activation markers is by flow cytometry, optionally by automated high-throughput flow cytometry, and optionally by an FX500 cell sorter. In some of the provided embodiments, one, two, three, or four runs of flow cytometry are performed to enrich for tumor-reactive T cells from the sample.

[0055] In some of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, and the sample is collected by needle biopsy, optionally by core needle biopsy or fine needle aspiration. In some embodiments, the biological sample comprises tumor fragments. In some of the provided embodiments, the T cell population comprises tumor-infiltrating lymphocytes.

[0056] Provided herein are methods for producing tumor-reactive T cells, the method comprising: (1) culturing the T cells by a process comprising: (a) incubating a T cell population derived from a biological sample from a subject with one or more recombinant cytokines to stimulate expansion of T cells in the population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; (b) enriching the T cell population for tumor-reactive T cells that contain an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a T cell population enriched for tumor-reactive T cells; and (c) incubating the T cell population enriched for tumor-reactive T cells with a T cell stimulator under conditions to stimulate expansion of cells of the cell population; and (2) harvesting the cells produced by the method.

[0057] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) culturing the T cells by a process comprising: (a) incubating a T cell population with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; (b) enriching the T cell population for tumor-reactive T cells that contain endogenous TCRs reactive to a tumor-associated antigen, thereby generating a T cell population enriched for tumor-reactive T cells; (c) incubating the T cell population enriched for tumor-reactive T cells with a T cell stimulator under conditions to stimulate expansion of cells of the cell population; and (d) further incubating the cells in the presence of one or more recombinant cytokines, optionally wherein the incubating in the presence of the one or more cytokines occurs before, during, and / or after the incubating with the T cell stimulator and / or the incubating in the presence of one or more T cell adjuvants, wherein the culturing stimulates expansion of cells of the T cell population; and (2) harvesting the cells produced by the method.

[0058] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) (a) incubating a cell population comprising T cells from a biological sample from a subject bearing a tumor with a T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant that is a checkpoint inhibitor; (b) incubating a cell population comprising T cells from a biological sample from a subject bearing a tumor with a T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population; (c) co-culturing the stimulated T cell population under conditions in which APCs are induced to express, thereby generating a population containing T cells, including tumor-reactive T cells; (c) enriching the co-culture for tumor-reactive T cells that contain endogenous TCRs reactive to the tumor-associated antigen, thereby generating a T cell population enriched for tumor-reactive T cells; (d) culturing the T cells by a process comprising incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of cells of the cell population; and (2) harvesting the expanded cells generated by the method.

[0059] In some of the provided embodiments, the at least one T cell adjuvant further comprises at least one apoptosis inhibitor. In some of the provided embodiments, the at least one apoptosis inhibitor and the checkpoint inhibitor are administered simultaneously, intermittently, or sequentially. In some of the provided embodiments, the at least one T cell adjuvant further comprises at least one costimulatory agonist. In some of the provided embodiments, the at least one costimulatory agonist and the checkpoint inhibitor are administered simultaneously, intermittently, or sequentially. In some of the provided embodiments, the apoptosis inhibitor inhibits caspase activation or activity.

[0060] In some of any of the provided embodiments, the apoptosis inhibitor inhibits one or more of caspase 2, caspase 8, caspase 9, caspase 10, caspase 3, caspase 6, or caspase 7. In some of the provided embodiments, the apoptosis inhibitor is selected from the group consisting of emricasan (IDN-6556, PF-03491390), NAIP (neuronal inhibitor of apoptosis protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome-linked IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8), wedelolactone, NS3694, NSCI, and Z-fluoromethylketone Z-VAD-FMK or a fluoromethylketone variant thereof. In some of the provided embodiments, the apoptosis inhibitor is a pan-caspase inhibitor that inhibits the activation or activity of two or more caspases. In some of the provided embodiments, the apoptosis inhibitor is Z-VAD-FMK. In some of the provided embodiments, the apoptosis inhibitor is selected from the group consisting of Z-FA-FMK, Z-VAD(OH)-FMK, Z-DEVD-FMK, Z-VAD(OM2)-FMK, and Z-VDVAD-FMK.

[0061] In some of any of the provided embodiments, the apoptosis inhibitor (e.g., a caspase inhibitor) is added at a concentration of about 0.5 μM to about 50 μM, about 0.5 μM to about 40 μM, about 0.5 μM to about 30 μM, about 0.5 μM to about 20 μM, about 0.5 μM to about 10 μM, about 0.5 μM to about 5 μM, about 0.5 μM to about 1 μM, about 1 μM to about 50 μM, about 1 μM to about 40 μM, about 1 μM to about 30 μM, about 1 μM to about 20 μM, about 1 μM to about 10 μM, or about 1 μM to about 5 μM, inclusive. In some embodiments, the apoptosis inhibitor (e.g., a caspase inhibitor) is added at a concentration of 2 μM or approximately 2 μM. In some embodiments, the apoptosis inhibitor (e.g., caspase inhibitor) is added at a concentration of 10 μM or about 10 μM. In some embodiments, the apoptosis inhibitor (e.g., caspase inhibitor) is added at a concentration of 25 μM or about 25 μM. In some of the provided embodiments, the apoptosis inhibitor (e.g., caspase inhibitor) is added at a concentration of from about 0.5 μg / mL to 25 μg / mL or about 25 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 10 μg / mL or about 10 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 5 μg / mL or about 5 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 1 μg / mL or about 1 μg / mL, from 1 μg / mL or about 1 μg / mL to 25 μg / mL. or from about 25 μg / mL, 1 μg / mL or about 1 μg / mL, 10 μg / mL or about 10 μg / mL, 1 μg / mL or about 1 μg / mL, 5 μg / mL or about 5 μg / mL, 5 μg / mL or about 5 μg / mL, 25 μg / mL or about 25 μg / mL, 5 μg / mL or about 5 μg / mL, 10 μg / mL or about 10 μg / mL, or 10 μg / mL or about 10 μg / mL, 25 μg / mL or about 25 μg / mL (inclusive).

[0062] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) (a) incubating a T cell population derived from a biological sample from a subject with one or more recombinant cytokines to stimulate expansion of T cells within the population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; (b) incubating in (a) in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens from the subject. (c) co-culturing the generated T cell population, thereby generating a population containing T cells, including tumor-reactive T cells; (c) enriching the tumor-reactive T cells generated in (b), thereby generating a T cell population enriched in tumor-reactive T cells, wherein the tumor-reactive T cells contain an endogenous TCR that is reactive to a tumor-associated antigen; (d) culturing the T cells by a process comprising incubating the tumor-reactive T cell-enriched T cell population with a T cell stimulator under conditions to stimulate expansion of cells of the cell population; and (2) harvesting the cells generated by the method.

[0063] In some of the provided embodiments, the method further comprises washing the cells after one or more of the steps of incubating the cells with a T cell stimulator, at least one T cell adjuvant, and / or one or more recombinant cytokines. In some of the provided embodiments, the culturing is carried out until a threshold amount of cells is obtained and / or for up to 30 days. In some of the provided embodiments, the culturing is carried out until a threshold amount of cells is obtained and / or for up to 20 days. In some of the provided embodiments, the T cell population comprises primary T cells from the subject, optionally a human subject. In some of the provided embodiments, the T cell population is isolated from an ex vivo co-culture comprising autologous T cells from a biological sample from the subject and antigen-presenting cells (APCs), the co-culture being carried out under conditions in which the APCs are induced to present one or more neo-antigenic peptides derived from a tumor from the subject. In some of the provided embodiments, the APCs are autologous to the subject or allogeneic to the subject. In some of the provided embodiments, the subject is healthy or not known to exhibit a disease or condition. In some of the provided embodiments, the T cell population is isolated or directly selected from a biological sample from the subject. In some of the provided embodiments, the subject exhibits a disease or condition, and optionally, the disease or condition is cancer. In some of the provided embodiments, the T cell population is allogeneic to the subject being treated or autologous to the subject being treated, and optionally, the subject being treated has a tumor or cancer.

[0064] In some of any of the provided embodiments, the biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample.

[0065] In some of the provided embodiments, the biological sample is a peripheral blood sample, and the peripheral blood sample is collected by blood draw or apheresis, and optionally, the apheresis is leukapheresis. In some of the provided embodiments, the biological sample has a volume of 50 mL to 400 mL. In some of the provided embodiments, enriching for tumor-reactive T cells or T cells surface-positive for one or more activation markers is by flow cytometry, optionally by automated high-throughput flow cytometry, and optionally by an FX500 cell sorter. In some of the provided embodiments, one, two, three, or four runs of flow cytometry are performed to enrich for tumor-reactive T cells from the sample.

[0066] In some of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, and the sample is collected by needle biopsy, optionally core needle biopsy, or fine needle aspiration. In some embodiments, the biological sample comprises tumor fragments. In some of the provided embodiments, the T cell population comprises tumor-infiltrating lymphocytes.

[0067] In some of any of the provided embodiments, enriching for tumor-reactive T cells comprises selecting for T cells that are surface positive for one or more T cell activation markers selected from the group consisting of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3.

[0068] In some of the provided embodiments, enriching for tumor-reactive T cells comprises selecting T cells that are surface-positive for one or more T cell activation markers. In some of the provided embodiments, the one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD69, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3. In some of the provided embodiments, the one or more T cell activation markers are selected from the group consisting of CD38, CD39, CD6, CD90, CD134, and CD137. In some of the provided embodiments, the one or more T cell activation markers are CD134 and / or CD137. In some of any of the provided embodiments, the one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, and CD256. In some of any of the provided embodiments, the one or more T cell activation markers are selected from the group consisting of CD107a, CD39, CD103, CD59, CD90, and CD38.

[0069] In some of any of the provided embodiments, the one or more T cell activation markers comprise at least two markers selected from CD107a and CD39, CD107a and CD103, CD107a and CD59, CD107a and CD90, CD107a and CD38, CD39 and CD103, CD39 and CD59, CD39 and CD90, CD39 and CD38, CD103 and CD59, CD103 and CD90, CD103 and CD38, CD59 and CD90, CD59 and CD38, and CD90 and CD38. In some of any of the provided embodiments, the one or more T cell activation markers further comprise CD137. In some of any of the provided embodiments, the one or more T cell activation markers comprise at least two markers selected from CD107a and CD137, CD38 and CD137, CD103 and CD137, CD59 and CD137, CD90 and CD137, and CD38 and CD 137. In some of any of the provided embodiments, the one or more T cell activation markers further comprise at least one marker selected from the group consisting of PD-1, TIM-3, and LAG-3.

[0070] In some of the provided embodiments, the T cells comprise CD4+ T cells, CD8+ T cells, or CD4+ T cells and CD8+ T cells. In some of the provided embodiments, the T cell population is enriched for CD4+ T cells and CD8+ T cells. In some of the provided embodiments, enriching for CD4+ T cells and CD8+ T cells comprises selecting for T cells that are surface positive for the cell surface marker CD3, or comprises sequential or simultaneous selection of T cells that are surface positive for the cell surface marker CD4 and T cells that are surface positive for the cell surface marker CD8. In some of any of the provided embodiments, the population enriched for CD4+ T cells and CD8+ T cells comprises greater than or about 60%, greater than or about 70%, greater than or about 80%, greater than or about 90%, or greater than or about 95% CD3+ T cells as a percentage of total cells in the population, or the population enriched for CD4+ T cells and CD8+ T cells comprises greater than or about 60%, greater than or about 70%, greater than or about 80%, greater than or about 80%, greater than or about 90%, or greater than or about 95% CD4+ T cells and CD8+ T cells as a percentage of total cells in the population. In some of any of the provided embodiments, the ratio of CD8+ T cells to CD4+ T cells is from at or about 1:100, 100:1 or about 100:1, 1:50 or about 1:50, 50:1 or about 50:1, 1:25 or about 1:25, 25:1 or about 25:1, 1:10 or about 1:10, 10:1 or about 10:1, 1:5 or about 1:5, 5:1 or about 5:1, or 1:2.5 or about 1:2.5, 2.5:1 or about 2.5:1.

[0071] In some of any of the provided embodiments, the tumor-associated antigen is a neo-antigen, and / or the tumor-reactive T cells comprise T cells comprising an endogenous TCR that is reactive to a neo-antigen, and in some of the provided embodiments, the population enriched for tumor-reactive T cells comprises greater than or about 60%, greater than or about 70%, greater than or about 70%, greater than or about 80%, greater than or about 80%, greater than or about 90%, or greater than or about 95% tumor-reactive T cells as a percentage of total cells or T cells in the population; and / or the population enriched for tumor-reactive T cells comprises greater than or about 60%, greater than or about 70%, greater than or about 70%, greater than or about 80%, greater than or about 80%, greater than or about 90%, or greater than or about 95% tumor-reactive T cells as a percentage of total cells or T cells in the population. The activated phenotype comprises T cells that are surface positive for one or more T cell activation markers selected from the group consisting of: CD107, CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3.

[0072] In some of any of the provided embodiments, the one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3. In some of any of the provided embodiments, the one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, and CD256. In some of any of the provided embodiments, the method includes selecting from the biological sample T cells that are surface positive for one or more T cell activation markers selected from the group consisting of CD107, CD107a, CD39, CD103, CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, and CD256.

[0073] Some of any of the provided embodiments further include incubating the selected cells in the presence of at least one T cell adjuvant and / or T cell stimulator under conditions for cell expansion or proliferation.

[0074] In some of the provided embodiments, the T cells are primary T cells from the subject, optionally a human subject. In some of the provided embodiments, the biological sample is an ex vivo co-culture comprising autologous T cells from the subject and antigen-presenting cells (APCs), wherein the co-culture is performed under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject. In some of the provided embodiments, the APCs are autologous to the subject or allogeneic to the subject. In some of the provided embodiments, the subject is healthy or not known to exhibit a disease or condition.

[0075] In some of the provided embodiments, the antigen-presenting cells are nucleated cells, such as dendritic cells, mononuclear phagocytes, B lymphocytes, endothelial cells, or thymic epithelium. In some of the provided embodiments, the antigen-presenting cells are dendritic cells. In some of the provided embodiments, the antigen-presenting cells are autologous to the subject or allogeneic to the subject. In some of the provided embodiments, the one or more peptides comprise at least one neoepitope derived from a tumor-associated antigen from the subject.

[0076] In some of the provided embodiments, the biological sample is obtained from a subject, and selecting includes isolating T cells directly from the biological sample. In some of the provided embodiments, the subject exhibits a disease or condition, and optionally, the disease or condition is cancer. In some of the provided embodiments, the subject has previously been administered a costimulatory agonist prior to isolating or selecting the T cell population. In some of the provided embodiments, the T cells are allogeneic to the subject being treated or autologous to the subject being treated, and optionally, the subject being treated has a tumor or cancer.

[0077] In some of the provided embodiments, the biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample. In some of the provided embodiments, the biological sample is a peripheral blood sample, and the peripheral blood sample is collected by blood draw or apheresis, and optionally, the apheresis is leukapheresis. In some of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, and the sample is collected by needle biopsy, optionally core needle biopsy, or fine needle aspiration.

[0078] In some of any of the provided embodiments, the one or more T cell activation markers are selected from the group consisting of CD107a, CD39, CD103, CD59, CD90, and CD38. In some of any of the provided embodiments, the one or more T cell activation markers comprise at least two markers selected from CD107a and CD39, CD107a and CD103, CD107a and CD59, CD107a and CD90, CD107a and CD38, CD39 and CD103, CD39 and CD59, CD39 and CD90, CD39 and CD38, CD103 and CD59, CD103 and CD90, CD103 and CD38, CD59 and CD90, CD59 and CD38, and CD90 and CD38.

[0079] In some of any of the provided embodiments, the one or more T cell activation markers further comprise CD 137. In some of any of the provided embodiments, the one or more reactive T cell activation markers comprise at least two markers selected from CD107a and CD137, CD38 and CD137, CD103 and CD137, CD59 and CD137, CD90 and CD137, and CD38 and CD137. In some of any of the provided embodiments, the one or more reactive T cell markers further comprise at least one marker selected from the group consisting of PD-1, TIM-3, and LAG-3.

[0080] In some of the provided embodiments, the selecting comprises immunoaffinity-based selection. In some of the provided embodiments, the immunoaffinity-based selection is performed by contacting the cells with an antibody that specifically binds to the marker and recovering the cells that bind to the antibody. In some of the provided embodiments, the antibody is immobilized on a particle, optionally the particle is a bead or nanoparticle, and optionally the particle is a magnetic particle. In some of the provided embodiments, the selecting comprises magnetic selection. In some of the provided embodiments, the antibody is labeled with a detectable agent, optionally the detectable agent is a fluorophore. In some of the provided embodiments, the selecting comprises flow cytometry.

[0081] In some of any of the provided embodiments, at least one T cell adjuvant is soluble and / or not bound or attached to a solid support, and optionally, the solid support is a bead.

[0082] In some of any of the provided embodiments, the at least one T cell adjuvant comprises at least one costimulatory agonist and at least one apoptosis blocker.

[0083] In some of any of the provided embodiments, the incubating with at least one costimulatory agonist and at least one apoptosis blocking agent is performed simultaneously, intermittently, or sequentially.

[0084] In some of the provided embodiments, the at least one T cell adjuvant comprises at least one costimulatory agonist. In some of the provided embodiments, the costimulatory agonist does not specifically bind to CD28 or is not an anti-CD28 antibody. In some of the provided embodiments, the costimulatory agonist is a tumor necrosis factor receptor superfamily (TNFRSF) agonist. In some of the provided embodiments, the costimulatory agonist is an antibody or antigen-binding fragment that specifically binds to a TNFRSF member, or a fusion protein comprising the extracellular domain of a ligand of a TNFRSF member or a binding portion thereof. In some of the provided embodiments, the TNFRSF member is selected from OX40, 4-1BB, GITR, and CD27.

[0085] In some of the provided embodiments, the costimulatory agonist specifically binds to OX40. In some of the provided embodiments, the costimulatory agonist is an antibody or antigen-binding fragment selected from Tavolixizumab, Pogalizumab, 11D4, 18D8, Hu119-122, Hu106-222, PF-04518600, GSK3174998, MEDI6469, BMS 986178, or 9B12, or is an antigen-binding fragment thereof. In some of the provided embodiments, the costimulatory agonist is an OX40L fusion protein that is MEDI6383. In some of the provided embodiments, the costimulatory agonist is Tavolixizumab.

[0086] In some of the provided embodiments, the costimulatory agonist specifically binds 4-1BB. In some of the provided embodiments, the costimulatory agonist is urelumab or utomilumab, or an antigen-binding fragment of any of the foregoing.

[0087] In some of any of the provided embodiments, the costimulatory agonist specifically binds CD27.

[0088] In some of the provided embodiments, the costimulatory agonist is varlilumab or any of the aforementioned antigen-binding fragments. In some of the provided embodiments, the costimulatory agonist specifically binds to GITR. In some of the provided embodiments, the costimulatory agonist is MK-1248 or any of the aforementioned antigen-binding fragments.

[0089] In some of the provided embodiments, the at least one T cell adjuvant comprises at least one apoptosis inhibitor. In some of the provided embodiments, the apoptosis inhibitor reduces apoptosis induced by CD95 (Fas), and optionally, the apoptosis inhibitor specifically binds to CD95 (Fas) or a CD95 ligand (Fas ligand). In some of the provided embodiments, the apoptosis inhibitor is an antibody or antigen-binding fragment, and optionally, the apoptosis inhibitor is an anti-Fas antibody or an anti-Fas ligand antibody. In some of the provided embodiments, the apoptosis inhibitor is a fusion protein comprising the extracellular domain of CD95 (Fas) or a specific-binding fragment thereof that binds to a CD95 ligand (Fas ligand) fused to an Fc immunoglobulin domain, and optionally, the apoptosis inhibitor is APG101 or CAN008.

[0090] In some of the provided embodiments, the apoptosis inhibitor inhibits activation or activity of a caspase, and optionally the caspase is caspase 2, caspase 8, caspase 9, caspase 10, caspase 3, caspase 6, or caspase 7, and optionally the caspase is caspase 3. In some of the provided embodiments, the apoptosis inhibitor is selected from the group consisting of NAIP (neuronal inhibitor of apoptosis protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome-linked IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7), and Ts-IAP (testis-specific IAP; BIRC8). In some of the provided embodiments, the apoptosis inhibitor is emericasan.

[0091] In some of any of the provided embodiments, the apoptosis inhibitor is present in a concentration of from 0.5 μg / mL or about 0.5 μg / mL to 25 μg / mL or about 25 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 10 μg / mL or about 10 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 5 μg / mL or about 5 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 1 μg / mL or about 1 μg / mL, from 1 μg / mL or about 1 μg / mL to 25 μg / mL. or about 25 μg / mL, from 1 μg / mL or about 1 μg / mL, from 10 μg / mL or about 10 μg / mL, from 1 μg / mL or about 1 μg / mL, from 5 μg / mL or about 5 μg / mL, from 5 μg / mL or about 5 μg / mL, from 25 μg / mL or about 25 μg / mL, from 5 μg / mL or about 5 μg / mL, from 10 μg / mL or about 10 μg / mL, and from 10 μg / mL or about 10 μg / mL, to 25 μg / mL or about 25 μg / mL, inclusive.

[0092] In some of the provided embodiments, the checkpoint inhibitor inhibits the activity of an immune checkpoint selected from the group consisting of PD-1 / PD-L1, CTLA-4, OX40, LAG-3, TIM-3, and B7-H3. In some of the provided embodiments, the immune checkpoint is selected from PD-1 / PD-L1. In some of the provided embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, optionally selected from pembrolizumab, cemiplimab, nivolumab, or an antigen-binding fragment of any of the foregoing. In some of the provided embodiments, the checkpoint inhibitor is pembrolizumab. In some of the provided embodiments, the checkpoint inhibitor is an anti-PDL1 antibody, optionally selected from avelumab, durvalumab, and atezolizumab, or an antigen-binding fragment of any of the foregoing.

[0093] In some of the provided embodiments, the immune checkpoint is OX40. In some of the provided embodiments, the checkpoint inhibitor is an anti-OX40L antibody, and optionally, the antibody is oxelumab or an antigen-binding fragment thereof.

[0094] In some of the provided embodiments, the immune checkpoint is CTLA-4. In some of the provided embodiments, the checkpoint inhibitor is an anti-CTLA-4 antibody, and optionally, the antibody is ipilimumab or an antigen-binding fragment thereof.

[0095] In some of any of the provided embodiments, the checkpoint inhibitor is administered at a concentration of from 0.5 μg / mL or about 0.5 μg / mL to 25 μg / mL or about 25 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 10 μg / mL or about 10 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 5 μg / mL or about 5 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 1 μg / mL or about 1 μg / mL, from 1 μg / mL or about 1 μg / mL to 25 μg / mL. or from about 25 μg / mL, 1 μg / mL or about 1 μg / mL, 10 μg / mL or about 10 μg / mL, 1 μg / mL or about 1 μg / mL, 5 μg / mL or about 5 μg / mL, 5 μg / mL or about 5 μg / mL, 25 μg / mL or about 25 μg / mL, 5 μg / mL or about 5 μg / mL, 10 μg / mL or about 10 μg / mL, and 10 μg / mL or about 10 μg / mL, 25 μg / mL or about 25 μg / mL (inclusive).

[0096] In some of any of the provided embodiments, the T cell adjuvant is added continuously during one or more stages of the culturing process, and the T cell adjuvant is replenished or replaced one or more times during the culturing process. In some of any of the provided embodiments, the T cell adjuvant is added continuously during incubation with one or more recombinant cytokines, and the T cell adjuvant is replenished or replaced one or more times during the incubation. In some of any of the provided embodiments, the T cell adjuvant is added transiently during one or more stages of the culturing process, and the T cell adjuvant is added only once during one or more stages of the culturing process.

[0097] In some of the provided embodiments, the T cell stimulatory agent is selected from an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling through a costimulatory receptor, optionally the costimulatory receptor being CD28. In some of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, optionally OKT3. In some of the provided embodiments, the agent that initiates signaling through a costimulatory receptor comprises peripheral blood mononuclear cells (PBMCs), optionally non-dividing PBMCs or irradiated PBMCs. In some of the provided embodiments, the agent that initiates signaling through a costimulatory receptor is an anti-CD28 antibody. In some of the provided embodiments, the T cell stimulatory agents are soluble anti-CD3 and anti-CD28 antibodies, respectively.

[0098] In some embodiments, the recombinant cytokine is selected from the group consisting of IL-10, IL-1a, IL-5, IL-7, IL-12, IL-23p40, IL-16, IL-17A, IL-15, IL-22, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL13, and IL-1b; optionally, the recombinant cytokine is selected from one or more of IL-2, IL-15, IL-7, and IL-21.

[0099] In some of any of the provided embodiments, the second T cell stimulator comprises a recombinant cytokine selected from the group consisting of IL-10, IL-1a, IL-5, IL-7, IL-12, IL-23p40, IL-16, IL-17A, IL-15, IL-22, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL13, and IL-1b.

[0100] In some of any of the provided embodiments, the T cell stimulator is or includes recombinant IL-2.

[0101] In some of any of the provided embodiments, the one or more recombinant cytokines include recombinant IL-2.

[0102] Provided herein is a method for producing tumor-reactive T cells, the method comprising: (1) (a) incubating a cell population comprising T cells from a biological sample obtained from a subject bearing a tumor with a first T cell stimulator comprising one or more recombinant cytokines selected from one or more of IL-7, IL-15, and IL-21 under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens from the subject, thereby generating a stimulated T cell population. (d) enriching the tumor-reactive T cell population reactive to one or more peptides from the co-culture, thereby producing a T cell population enriched in tumor-reactive T cells, wherein the tumor-reactive T cells comprise an endogenous TCR reactive to a tumor-associated antigen; and (d) culturing the T cells by a process comprising incubating the tumor-reactive T cell-enriched T cell population with a second T cell stimulator under conditions to stimulate expansion of T cells within the population, and (2) harvesting the expanded cells produced by the method.

[0103] In some of any of the provided embodiments, the first T cell stimulator comprises one or more recombinant cytokines including IL-7 and IL-15. In some of the provided embodiments, the first T cell stimulator further comprises recombinant IL-2. In some of the provided embodiments, the first T cell stimulator does not comprise recombinant IL-2.

[0104] In some of any of the provided embodiments, the second T cell stimulator comprises one or more recombinant cytokines selected from the group consisting of IL-10, IL-1a, IL-5, IL-7, IL-12, IL-23p40, IL-16, IL-17A, IL-15, IL-22, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL13, and IL-1b. In some of any of the provided embodiments, the recombinant cytokine is selected from one or more of IL-2, IL-15, IL-7, and IL-21. In some of any of the provided embodiments, the one or more recombinant cytokines comprises recombinant IL-2.

[0105] In some of any of the provided embodiments, the concentration of each of the one or more recombinant cytokines individually is between 100 IU / mL and 6000 IU / mL. In some of the provided embodiments, the concentration of each of the one or more recombinant cytokines individually is between 300 IU / mL and 1000 IU / mL, and optionally, the concentration of each of the one or more recombinant cytokines is at or about 300 IU / mL, or 1000 IU / mL or about 1000 IU / mL.

[0106] In some of any of the provided embodiments, the second T cell stimulator further comprises an anti-CD3 antibody, optionally OKT3, and optionally the concentration of the anti-CD3 antibody is at or about 50 ng / mL.

[0107] In some of any of the provided embodiments, the co-culturing is carried out by a process comprising: (a) identifying somatic mutations associated with one or more tumor-associated antigens by exome sequencing of healthy and tumor tissues from the subject; (b) identifying at least one neoepitope of the one or more tumor-associated antigens; (c) isolating an autologous T cell population from a biological sample from the subject; and (d) co-culturing the T cell population with antigen-presenting cells (APCs) that have been exposed to or contacted with one or more peptides comprising at least one neoepitope of the one or more tumor-associated antigens under conditions for presenting one or more of the peptides on the surface of a major histocompatibility complex (MHC), thereby generating a T cell population comprising tumor-reactive T cells that are reactive to the one or more peptides.

[0108] In some of the embodiments provided, the MHC molecule is a class I molecule. In some of the embodiments provided, the MHC molecule is a class II molecule. In some of the embodiments provided, the MHC molecule is MHC class I and II.

[0109] In some of the provided embodiments, the T cells are CD4+ cells. In some of the provided embodiments, the T cells are CD8+ cells. In some of the provided embodiments, the T cells are CD4+ and CD8+ cells.

[0110] In some of the provided embodiments, the one or more peptides comprise individual peptides or peptide pools. In some of the provided embodiments, the one or more peptides are loaded onto antigen-presenting cells by transfection of an in vitro transcribed synthetic minigene construct encoding the one or more peptides, optionally flanked in tandem by 12 amino acids from an endogenous protein, and the transcription minigene construct generates the individual peptides. In some of the provided embodiments, the one or more peptides are loaded onto antigen-presenting cells by peptide pulsing, optionally by electroporation. In some of the provided embodiments, the one or more peptides are each individually 5-30 amino acids in length, optionally 12-25 amino acids in length, optionally 25 amino acids in length, or about 25 amino acids in length.

[0111] In some of any of the provided embodiments, the one or more peptides are a peptide pool and the concentration of the peptides in the peptide pool for the peptide pulse is from 0.001 μg / mL or about 0.001 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL to 40 μg / mL or about 40 μg / mL, 0.1 μg / mL or about 0.1 μg / mL to 40 μg / mL or about 40 μg / mL, 1 μg / mL or about 1 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL or about 0.01 μg / mL to 10 μg / mL or about 10 μg / mL, or 1 μg / mL or about 1 μg / mL to 10 μg / mL or about 10 μg / mL, or the one or more peptides are individual peptides and the concentration of the individual peptides for the peptide pulse is from 0.01 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL to 40 μg / mL or about 40 μg / mL, 0.1 μg / mL to 40 μg / mL or about 40 μg / mL, 0.1 μg / mL to 40 μg / mL or about 40 μg / mL, 0.1 μg / mL to 40 μg / mL or about 1 μg / mL to 10 μg / mL or about 10 μg / mL The concentration of is from 0.00001 μg / mL or about 0.00001 μg / mL to 1 μg / mL or about 1 μg / mL, 0.00001 μg / mL or about 0.00001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, 0.00001 μg / mL or about 0.00001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL, 0.0001 μg / mL or about 0.0001 μg / mL In some of the provided embodiments, the concentration of an individual peptide of the one or more peptides is, on average, from 0.00001 μg / mL or about 0.00001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL. In some of any of the provided embodiments, the concentration of an individual peptide of the one or more peptides is, on average, at or about 0.0001 μg / mL to at or about 0.001 μg / mL.

[0112] In some of any of the provided embodiments, the harvesting step is performed within 20 days after initiation of culturing and / or enriching the T cells, including tumor-reactive cells. In some of the provided embodiments, the culturing step is performed in the presence of a recombinant cytokine selected from the group consisting of IL-2, IL-15, IL-7, and IL-21.

[0113] In some of any of the provided embodiments, the cells are harvested 7 to 20 days, 7 to 14 days, 7 to 10 days, 10 to 20 days, 10 to 14 days, or 14 to 20 days after initiation of culture.

[0114] In some of the provided embodiments, the culturing step comprises culturing 0.5 x 10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 0.5 x 10 total cells or total viable cells 8 From 12 x 10 9 pieces or approximately 12 x 10 9 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 3.5 x 10 8pieces or approximately 3.5 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 1 x 10 whole cells or total viable cells 8 From 50 x 10 9 or approximately 50 x 10 9 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 1 x 10 whole cells or total viable cells 8 From 12 x 10 9 pieces or approximately 12 x 10 9 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 93.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 50 x 10 9or approximately 50 x 10 9 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 60 x 10 whole cells or total viable cells 8 pieces or approximately 60 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 60 x 10 whole cells or total viable cells 8 pieces or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 60 x 10 whole cells or total viable cells 8 pieces or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 12 x 10 total cells or total viable cells 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 12 x 10 total cells or total viable cells 9 pieces or approximately 12 x 10 9 From 30 x 10 9 pieces or approximately 30 x 10 9 total or viable cells, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 pieces or approximately 60 x 10 9 This is done until a threshold amount of cells is achieved, which is either total cells or total viable cells (inclusive).

[0115] In some of any of the provided embodiments, the method results in a fold expansion of T cells or a fold expansion of tumor-reactive T cells that is at least or at least about 2-fold, at least or at least about 5-fold, at least or at least about 10-fold, at least or at least about 25-fold, at least or at least about 25-fold, at least or at least about 50-fold, at least or at least about 100-fold, at least or at least about 250-fold, at least or at least about 500-fold, at least or at least about 1000-fold, or more.

[0116] In some of any of the provided embodiments, the biological sample is a peripheral blood sample, optionally an apheresis sample, and the number of cells at the start of the culture is 1 x 10 9 pieces or approximately 1 x 10 9 From 7 x 10 9 total viable cells; or 1 x 10 9 or approximately 1 x 10 9 Total viable cells, 2 x 10 9 or approximately 2 x 10 9 Total viable cells, 3 x 10 9 Total viable cells, 4 x 10 9 Total viable cells, 5 x 10 9 Total viable cells, 6 x 10 9 total viable cells, or 7 x 10 9 and / or the percentage of tumor-reactive T cells at the initiation of the culture is from 0.02% or about 0.02% to 40% or about 40%, from 0.02% or about 0.02% to 24% or about 24%, from 0.02% or about 0.02% to 18% or about 18%, from 0.02% or about 0.02% to 0.9% or about 0.9%, or from 0.02% or about 0.02% to 6.0% or about 6.0%; and / or the number of T cells surface-positive for a T cell activation marker at the initiation of the culture is 0.1 x 10 6 or approximately 0.1 x 10 6 From 60 x 10 6pieces or approximately 60 x 10 6 T cells, 0.1 x 10 6 From 8 x 10 6 pieces or approximately 8 x 10 6 T cells, 0.1 x 10 6 From 20 x 10 6 pieces or approximately 20 x 10 6 T cells, 0.3 x 10 6 From 35 x 10 6 pieces or approximately 35 x 10 6 T cells, or 0.3 x 10 6 From 60 x 10 6 pieces or approximately 60 x 10 6 or 0.1 x 10 T cells 6 T cells, 0.3 x 10 6 T cells, 0.6 x 10 6 T cells, 1 x 10 6 T cells, 5 x 10 6 T cells, 10 x 10 6 T cells, 35 x 10 6 T cells, or 60 x 10 6 T cells or approximately 0.1 x 10 6 T cells, 0.3 x 10 6 T cells, 0.6 x 10 6 T cells, 1 x 10 6 T cells, 5 x 10 6 T cells, 10 x 10 6 T cells, 35 x 10 6 T cells, or 60 x 10 6 T cells, or any value between any of the aforementioned.

[0117] In some of any of the provided embodiments, the biological sample is a lymphoid-derived sample or a tumor-derived sample, and the number of cells at the start of the culture is 10 x 10 6 pieces or approximately 10 x 10 6 From 100 x 10 6 Total viable cells, 20 x 10 6 From 100 x 10 6 total viable cells, or 12 x 10 6 From 43 x 10 6total viable cells; or 10 x 10 6 pieces or approximately 10 x 10 6 Total viable cells, 12 x 10 6 pieces or approximately 12 x 10 6 Total viable cells, 20 x 10 6 Total viable cells, 40 x 10 6 Total viable cells, 60 x 10 6 total viable cells, or 100 x 10 6 total viable cells, or any value between any of the foregoing; and / or the percentage of tumor-reactive T cells at the initiation of the culture is from 1% or about 1% to 90% or about 90%, from 1% or about 1% to 75% or about 75%, from 1% or about 1% to 50% or about 50%, from 1% or about 1% to 25% or about 25%, or from 1% or about 1% to 14% or about 14%; and / or the number of T cells surface-positive for a T cell activation marker at the initiation of the culture is 0.7 x 10 6 or approximately 0.7 x 10 6 From 15 x 10 6 pieces or approximately 15 x 10 6 T cells, 1 x 10 6 From 15 x 10 6 pieces or approximately 15 x 10 6 T cells, or 0.7 x 10 6 or approximately 0.7 x 10 6 From 5.4 x 10 6 or approximately 5.4 x 10 6 or 0.7 x 10 T cells 6 T cells, 1 x 10 6 T cells, 5.4 x 10 6 T cells, or 15 x 10 6 T cells or approximately 0.7 × 10 6 T cells, 1 x 10 6 T cells, 5.4 x 10 6 T cells, or 15 x 10 6 T cells, or any value between any of the aforementioned.

[0118] In some of any of the provided embodiments, the cell population comprising T cells comprises tumor-infiltrating lymphocytes, lymph lymphocytes, or peripheral blood mononuclear cells.

[0119] In some of the provided embodiments, the biological sample is a tumor and the cell population comprising T cells comprises tumor-infiltrating lymphocytes. In some of the provided embodiments, the biological sample is a resected tumor and the cell population comprising T cells is one or more tumor fragments from the resected tumor. In some of the provided embodiments, the one or more tumor fragments are about 1 tumor fragment / 2 cm. 2 and seeded for incubation with the first T cell stimulator. In some of the provided embodiments, the tumor is melanoma. In some of the provided embodiments, the biological sample is a resected tumor and the cell population comprising T cells is processed as a single cell suspension by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor. In some of the provided embodiments, the biological sample is a resected tumor and the cell population comprising T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from the resected tumor. In some of the provided embodiments, the enzymatic digestion is by incubation with collagenase, optionally collagenase IV or collagenase I / II.

[0120] In some of any of the provided embodiments, the cell population comprising T cells is between about 5 x 10 to 2 x 10 or about 2 x 10 total cells / 2 cm. 2 and seeded for incubation with the first T cell stimulator. In some of any of the provided embodiments, the tumor is colorectal cancer (CRC).

[0121] In some of any of the provided embodiments, incubation with at least one T cell adjuvant is for a minimum of 24 hours up to the time of cell harvest, or from at or about 24 hours to at or about 96 hours, or from at or about 24 hours to at or about 48 hours, or for at or about 24 hours, 36 hours, or about 36 hours, or 48 hours, or about 48 hours.

[0122] In some of any of the provided embodiments, incubation with the T cell stimulator is for at or about 24 hours to at or about 96 hours, or from at or about 24 hours to at or about 48 hours, or for at or about 24 hours, 36 hours, or about 36 hours, or 48 hours, or about 48 hours.

[0123] In some of the provided embodiments, incubating with the first T cell stimulator is for 7 to 21 days, optionally 7 to 14 days. In some of the provided embodiments, incubating with the first T cell stimulator is in a closed system. In some of the provided embodiments, incubating with the first T cell stimulator is in a gas-permeable culture vessel. In some of the provided embodiments, incubating with the first T cell stimulator is performed using a bioreactor. In some of the provided embodiments, incubating with the second T cell stimulator is for 7 to 21 days, optionally 7 to 14 days. In some of the provided embodiments, incubating with the second T cell stimulator is in a closed system. In some of the provided embodiments, incubating with the second T cell stimulator is in a gas-permeable culture vessel. In some of the provided embodiments, incubating with the second T cell stimulator is performed using a bioreactor.

[0124] In some of any of the provided embodiments, one or more of the method steps are performed in a closed system, and optionally one or more of the selection and / or enrichment or incubation steps are performed in a closed system.

[0125] In some of the provided embodiments, the method further comprises formulating the harvested cells for administration to the subject, hi some of the provided embodiments, the formulating comprises cryopreservation, and the cells are thawed prior to administration to the subject.

[0126] Provided herein are compositions produced by any of the provided methods.

[0127] Provided herein are compositions comprising tumor-reactive T cells, wherein at least 40%, or at least about 40%, at least 50%, or at least about 50%, at least 60%, or at least about 60%, at least 70%, or at least about 70%, at least 80%, or at least about 80%, or at least 90% or at least about 90% of the total cells or T cells in the composition are tumor-reactive T cells or are surface positive for one or more T cell activation markers.

[0128] Provided herein are compositions comprising expanded T cells enriched for tumor-reactive T cells produced by any of the methods provided.

[0129] Provided herein are compositions comprising a pharmaceutically acceptable excipient produced by any of the provided methods.

[0130] In some of the embodiments of the provided compositions, the one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3. In some embodiments, the one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, and CD256. In some embodiments, the one or more T cell activation markers are selected from the group consisting of CD107a, CD39, CD103, CD59, CD90, and CD38. In some embodiments, the one or more T cell activation markers comprise at least two markers selected from CD107a and CD39, CD107a and CD103, CD107a and CD59, CD107a and CD90, CD107a and CD38, CD39 and CD103, CD39 and CD59, CD39 and CD90, CD39 and CD38, CD103 and CD59, CD103 and CD90, CD103 and CD38, CD59 and CD90, CD59 and CD38, and CD90 and CD38. In some embodiments, the one or more T cell activation markers further comprise CD137. In some embodiments, the one or more reactive T cell activation markers comprise at least two markers selected from CD107a and CD137, CD38 and CD137, CD103 and CD137, CD59 and CD137, CD90 and CD137, and CD38 and CD 137. In some embodiments, the one or more reactive T cell markers further comprise at least one marker selected from the group consisting of PD-1, TIM-3, and LAG-3.

[0131] In some embodiments of any of the provided compositions, the T cells are CD3+ T cells, or comprise CD4+ T cells and / or CD8+ T cells. In some embodiments, the T cells comprise CD4+ T cells and CD8+ T cells, and the ratio of CD8+ T cells to CD4+ T cells is from at or about 1:100 to 100:1 or about 100:1, 1:50 or about 1:50 to 50:1 or about 50:1, 1:25 or about 1:25 to 25:1 or about 25:1, 1:10 or about 1:10 to 10:1 or about 10:1, 1:5 or about 1:5 to 5:1 or about 5:1, or 1:2.5 or about 1:2.5 to 2.5:1 or about 2.5:1.

[0132] In some of any of the provided embodiments, the method results in a fold expansion of T cells or a fold expansion of tumor-reactive T cells that is at least or at least about 2-fold, at least or at least about 5-fold, at least or at least about 10-fold, at least or at least about 25-fold, at least or at least about 25-fold, at least or at least about 50-fold, at least or at least about 100-fold, at least or at least about 250-fold, at least or at least about 500-fold, at least or at least about 1000-fold, or more.

[0133] In some of any of the provided embodiments, the composition of expanded cells is capable of producing IFNγ after antigen-specific stimulation at a concentration of greater than or about 30 pg / mL, optionally greater than or about 60 pg / mL. In some of any of the provided embodiments, the method includes formulating the harvested cells with a cryoprotectant.

[0134] In some of the provided embodiments, the T cells are CD3+ T cells or comprise CD4+ T cells and / or CD8+ T cells. In some of the provided embodiments, the T cells comprise CD4+ T cells and CD8+ T cells, and the ratio of CD8+ T cells to CD4+ T cells is from at or about 1:100 to 100:1 or about 100:1, 1:50 or about 1:50 to 50:1 or about 50:1, 1:25 or about 1:25 to 25:1 or about 25:1, 1:10 or about 1:10 to 10:1 or about 10:1, 1:5 or about 1:5 to 5:1 or about 5:1, or 1:2.5 or about 1:2.5 to 2.5:1 or about 2.5:1.

[0135] In some of any of the provided embodiments, the number of tumor-reactive T cells in the composition, or the total number of T cells surface-positive for a T cell activation marker, or the number of viable cells thereof, is 0.5 x 10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 0.5×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 pieces, 1×10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 1×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 3.5×108 pieces or approximately 3.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 15×10 8pieces or approximately 15 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 pieces or approximately 60 x 10 9 (inclusive).

[0136] In some of the embodiments of the provided compositions, the number of tumor-reactive T cells in the composition, or the total number of T cells surface-positive for a T cell activation marker, or the number of viable cells thereof, is 0.5 x 10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 0.5×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 pieces, 1×10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 1×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 3.5 x 10 8pieces or approximately 3.5 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 15 x 10 8pieces or approximately 15 x 10 8 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 pieces or approximately 60 x 10 9 (inclusive).

[0137] In some of any of the provided embodiments, the therapeutically effective dose is 1×10 9 ~10×10 9 These are T cells.

[0138] In some of the embodiments of the provided compositions, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the composition comprises a cryoprotectant.

[0139] In some of any of the embodiments of the compositions provided, the compositions are sterile.

[0140] Provided herein are methods of treatment that include administering to a subject having a tumor a therapeutic dose of a composition produced by any of the provided methods.

[0141] Provided herein is a method of treatment comprising administering any of the provided compositions to a subject with cancer. In some embodiments, the cells of the administered composition are autologous to the subject. In some embodiments, the cancer is epithelial cancer.

[0142] In some embodiments, the cancer is melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer.

[0143] In some of the provided embodiments, a composition comprising the expanded cells produced by the method is used to treat cancer in a subject. In some of the provided embodiments, the tumor is an epithelial cancer tumor. In some of the provided methods, the tumor is a melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, gastric cancer, or uterine cancer tumor. In some of the provided methods, the tumor is a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer tumor, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer. [The present invention 1001] 1. A method for producing tumor-reactive T cells, comprising: (1)(a) incubating a cell population comprising T cells from a biological sample obtained from a subject having a tumor with a first T cell stimulator under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a population containing T cells, including tumor-reactive T cells; (d) enriching the co-culture for a tumor-reactive T cell population that is reactive to the one or more peptides, thereby producing a T cell population enriched for tumor-reactive T cells, wherein the tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen; and (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of T cells within the population. Culturing T cells by a process comprising: one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant that is an apoptosis inhibitor that inhibits caspase activation or activity; (2) harvesting the cells produced by the method to produce a composition of expanded T cells enriched for tumor-reactive T cells; The method comprises: [The present invention 1002] The method of claim 1001, wherein said first T cell stimulator or second T cell stimulator comprises the presence of one or more recombinant cytokines, and wherein incubation in the presence of said T cell stimulator is performed before, during, and / or after incubation in the presence of one or more of said T cell adjuvants. [The present invention 1003] 1002. The method of claim 1001, wherein said incubation with at least one T cell adjuvant occurs during at least a portion of said co-culture. [The present invention 1004] Any of the methods of inventions 1001 to 1003, wherein at least a portion of the incubating of the T cell population with the at least one T cell adjuvant is performed simultaneously with incubating the T cell population with the T cell stimulator. [The present invention 1005] 1. A method for producing tumor-reactive T cells, comprising: (1)(a) incubating a cell population comprising T cells from a biological sample from a subject having a tumor with a first T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant that is an apoptosis inhibitor that inhibits caspase activation or activity; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a population containing T cells, including tumor-reactive T cells; (c) enriching the co-culture for tumor-reactive T cells that contain endogenous TCRs reactive to tumor-associated antigens, thereby generating a T cell population enriched for tumor-reactive T cells; (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate cellular expansion of the cells of the cell population. Culturing the T cells by a process comprising: (2) harvesting the cells produced by the method; The method comprises: [The present invention 1006] 1006. The method of any of claims 1001 to 1005, wherein said at least one T cell adjuvant further comprises at least one costimulatory agonist. [The present invention 1007] 1006. The method of claim 1006, wherein said incubating with said at least one costimulatory agonist and said apoptosis-inhibiting substance is performed simultaneously, intermittently, or sequentially. [The present invention 1008] 1006. The method of any of claims 1001 to 1005, wherein said at least one T cell adjuvant further comprises at least one checkpoint inhibitor. [The present invention 1009] 1009. The method of claim 8, wherein said incubating with said at least one checkpoint inhibitor and said apoptosis inhibitor is performed simultaneously, intermittently, or sequentially. [The present invention 1010] 1. A method for producing tumor-reactive T cells, comprising: (1)(a) incubating a cell population comprising T cells from a biological sample obtained from a subject having a tumor with a first T cell stimulator under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a population containing T cells, including tumor-reactive T cells; (d) enriching the co-culture for a tumor-reactive T cell population that is reactive to the one or more peptides, thereby producing a T cell population enriched for tumor-reactive T cells, wherein the tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen; and (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of T cells within the population. Culturing T cells by a process comprising: one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant that is a costimulatory agonist; (2) harvesting the cells produced by the method to produce a composition of expanded T cells enriched for tumor-reactive T cells; The method comprises: [The present invention 1011] The method of claim 1010, wherein said first T cell stimulator or second T cell stimulator comprises the presence of one or more recombinant cytokines, and wherein incubation in the presence of said T cell stimulator is performed before, during, and / or after incubation in the presence of one or more of said T cell adjuvants. [The present invention 1012] 10. The method of claim 10, wherein said incubation with at least one T cell adjuvant occurs during at least a portion of said co-culture. [The present invention 1013] Any of the methods of claims 1010 to 1012, wherein at least a portion of the incubating of the T cell population with the at least one T cell adjuvant is performed simultaneously with incubating the T cell population with the T cell stimulator. [The present invention 1014] 1. A method for producing tumor-reactive T cells, comprising: (1)(a) incubating a cell population comprising T cells from a biological sample from a subject having a tumor with a first T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant that is a costimulatory agonist; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a population containing T cells, including tumor-reactive T cells; (c) enriching the co-culture for tumor-reactive T cells that contain endogenous TCRs reactive to tumor-associated antigens, thereby generating a T cell population enriched for tumor-reactive T cells; (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate cellular expansion of the cells of the cell population. Culturing the T cells by a process comprising: (2) harvesting the expanded cells produced by the method; The method comprises: [The present invention 1015] 1015. The method of any of claims 1010 to 1014, wherein said at least one T cell adjuvant further comprises at least one apoptosis inhibitor. [The present invention 1016] 1015. The method of claim 1015, wherein incubating with said at least one apoptosis inhibitor and said checkpoint agonist is performed simultaneously, intermittently, or sequentially. [The present invention 1017] The method of any of claims 1010 to 1014, wherein said at least one T cell adjuvant further comprises at least one checkpoint inhibitor. [The present invention 1018] 1018. The method of claim 1017, wherein incubating with said at least one checkpoint inhibitor and said costimulatory agonist is performed simultaneously, intermittently, or sequentially. [The present invention 1019] 1. A method for producing tumor-reactive T cells, comprising: (1)(a) incubating a cell population comprising T cells derived from a biological sample from a subject having a tumor with a first T cell stimulator under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a population containing T cells, including tumor-reactive T cells; (d) enriching the co-culture for a tumor-reactive T cell population that is reactive to the one or more peptides, thereby producing a T cell population enriched for tumor-reactive T cells, wherein the tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen; and (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of T cells within the population. Culturing T cells by a process comprising: one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant that is a checkpoint inhibitor; (2) harvesting the expanded cells produced by the method; The method comprises: [The present invention 1020] The method of claim 1019, wherein said first T cell stimulator or second T cell stimulator comprises the presence of one or more recombinant cytokines, and wherein incubation in the presence of said T cell stimulator is performed before, during, and / or after incubation in the presence of one or more of said T cell adjuvants. [The present invention 1021] 1019. The method of claim 1019, wherein said incubation with at least one T cell adjuvant occurs during at least a portion of said co-culture. [The present invention 1022] Any of the methods of claims 1019 to 1021, wherein at least a portion of the incubating of the T cell population with the at least one T cell adjuvant is performed simultaneously with incubating the T cell population with the T cell stimulator. [The present invention 1023] 1. A method for producing tumor-reactive T cells, comprising: (1)(a) incubating a cell population comprising T cells from a biological sample from a subject having a tumor with a T cell stimulator comprising one or more recombinant cytokines under conditions to stimulate expansion of T cells within the population to generate a stimulated T cell population, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvant that is a checkpoint inhibitor; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a population containing T cells, including tumor-reactive T cells; (c) enriching the co-culture for tumor-reactive T cells that contain endogenous TCRs reactive to tumor-associated antigens, thereby generating a T cell population enriched for tumor-reactive T cells; (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate cellular expansion of the cells of the cell population. Culturing the T cells by a process comprising: (2) harvesting the expanded cells produced by the method; The method comprises: [The present invention 1024] 1024. The method of any of claims 1019 to 1023, wherein said at least one T cell adjuvant further comprises at least one apoptosis inhibitor. [The present invention 1025] 1025. The method of claim 1024, wherein said incubating with said at least one apoptosis inhibitor and said checkpoint inhibitor is performed simultaneously, intermittently, or sequentially. [The present invention 1026] 1024. The method of any of claims 1019 to 1023, wherein said at least one T cell adjuvant further comprises at least one costimulatory agonist. [The present invention 1027] 1027. The method of claim 1026, wherein said incubating with said at least one costimulatory agonist and said checkpoint inhibitor is performed simultaneously, intermittently, or sequentially. [The present invention 1028] The method of any of claims 1015, 1016, 1024, and 1025, wherein said apoptosis inhibitor inhibits the activation or activity of a caspase. [The present invention 1029] 1029. The method of any of claims 1001 to 1009, 1015, 1016, 1024, 1025, and 1028, wherein the apoptosis inhibitor inhibits one or more of caspase 2, caspase 8, caspase 9, caspase 10, caspase 3, caspase 6, or caspase 7. [The present invention 1030] Any of the methods of inventions 1001 to 1009, 1015, 1016, 1024, 1025, 1028, and 1029, wherein the apoptosis inhibitor is selected from the group consisting of emricasan (IDN-6556, PF-03491390), NAIP (neuronal inhibitor of apoptosis protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome-linked IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8), wedelolactone, NS3694, NSCI, and Z-fluoromethylketone Z-VAD-FMK or a fluoromethylketone variant thereof. [The present invention 1031] The method of any one of claims 1001 to 1009, 1015, 1016, 1024, 1025, and 1028 to 1030, wherein the apoptosis inhibitor is a pan-caspase inhibitor that inhibits the activation or activity of two or more caspases. [The present invention 1032] The method of any one of claims 1001 to 1009, 1015, 1016, 1024, 1025, and 1028 to 1031, wherein the apoptosis inhibitor is Z-VAD-FMK. [The present invention 1033] Any of the methods of present inventions 1001 to 1009, 1015, 1016, 1024, 1025, and 1028 to 1032, wherein the apoptosis inhibitor is selected from the group consisting of Z-FA-FMK, Z-VAD(OH)-FMK, Z-DEVD-FMK, Z-VAD(OM2)-FMK, and Z-VDVAD-FMK. [The present invention 1034] The apoptosis inhibitor is from about 0.5 μM to about 50 μM, from about 0.5 μM to about 40 μM, from about 0.5 μM to about 30 μM, from about 0.5 μM to about 20 μM, from about 0.5 μM to about 10 μM, from about 0.5 μM to about 5 μM, from about 0.5 μM to about 1 μM, from about 1 μM to about 50 μM, from about 1 μM to about 40 μM, from about 1 μM to about 30 μM, from about 1 μM to about 5 μM, 10 μM to about 20 μM, about 1 μM to about 10 μM, about 1 μM to about 5 μM (inclusive), and optionally, the concentration is 2 μM or about 2 μM, 10 μM or about 10 μM, or 25 μM or about 25 μM. [This invention 1035] The apoptosis inhibitor is from about 0.5 μg / mL to 25 μg / mL or about 25 μg / mL, 0.5 μg / mL or about 0.5 μg / mL to 10 μg / mL or about 10 μg / mL, 0.5 μg / mL or about 0.5 μg / mL to 5 μg / mL or about 5 μg / mL, 0.5 μg / mL or about 0.5 μg / mL to 1 μg / mL or about 1 μg / mL, 1 μg / mL or about 1 μg / mL to 25 μg / mL or about 25 μg / mL, 1 μg / mL or about 1 μg / mL to 10 μg / mL or Any of the methods of inventions 1001 to 1009, 1015, 1016, 1024, 1025, and 1028 to 1033, wherein the α-glucan is added at a concentration of from about 10 μg / mL, 1 μg / mL or about 1 μg / mL, 5 μg / mL or about 5 μg / mL, 5 μg / mL or about 5 μg / mL, 25 μg / mL or about 25 μg / mL, 5 μg / mL or about 5 μg / mL, 10 μg / mL or about 10 μg / mL, or 10 μg / mL or about 10 μg / mL, 25 μg / mL or about 25 μg / mL (inclusive). [The present invention 1036] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, and 1027, wherein the costimulatory agonist is a tumor necrosis factor receptor superfamily (TNFRSF) agonist. [This invention 1037] Any of methods 1006, 1007, 1010 to 1018, 1026, 1027, and 1036, wherein the costimulatory agonist is an antibody or antigen-binding fragment that specifically binds to a TNFRSF member, or a fusion protein comprising the extracellular domain of a ligand of the TNFRSF member or a binding portion thereof. [The present invention 1038] 1036. The method of claim 1036, wherein said TNFRSF member is selected from OX40, 4-1BB, GITR, and CD27. [This invention 1039] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, 1027, and 1036 to 1038, wherein the costimulatory agonist specifically binds to OX40. [The present invention 1040] Any of the methods of claims 1006, 1007, 1010 to 1018, 1026, 1027, and 1036 to 1039, wherein the costimulatory agonist is an antibody or antigen-binding fragment selected from tabolixizumab, pogalizumab, 11D4, 18D8, Hu119-122, Hu106-222, PF-04518600, GSK3174998, MEDI6469, BMS 986178, or 9B12, or an antigen-binding fragment thereof. [The present invention 1041] 1040. The method of claim 1040, wherein said costimulatory agonist is tabolixizumab. [The present invention 1042] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, 1027, and 1036 to 1038, wherein the costimulatory agonist specifically binds to 4-1BB. [This invention 1043] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, 1027, 1036 to 1038, and 1042, wherein the costimulatory agonist is urelumab or utomilumab, or an antigen-binding fragment of any of the foregoing. [This invention 1044] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, 1027, and 1036 to 1038, wherein the costimulatory agonist specifically binds to CD27. [This invention 1045] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, 1027, 1036 to 1038, and 1044, wherein the costimulatory agonist is varlilumab or an antigen-binding fragment of any of the foregoing. [The present invention 1046] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, 1027, and 1036 to 1038, wherein the costimulatory agonist specifically binds to GITR. [This invention 1047] The method of any one of claims 1006, 1007, 1010 to 1018, 1026, 1027, 1036 to 1038, and 1046, wherein the costimulatory agonist is MK-1248 or any of the aforementioned antigen-binding fragments. [This invention 1048] The costimulatory agonist is from 0.5 μg / mL or about 0.5 μg / mL to 25 μg / mL or about 25 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 10 μg / mL or about 10 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 5 μg / mL or about 5 μg / mL, from 0.5 μg / mL or about 0.5 μg / mL to 1 μg / mL or about 1 μg / mL, from 1 μg / mL or about 1 μg / mL to 25 μg / mL or about 25 μg / mL, from 1 μg / mL or about 1 μg / mL to 10 μg / mL Any of methods 1006, 1007, 1010 to 1018, 1026, 1027, 1036 to 1047, wherein the hydroxybenzoate is added at a concentration of from 1 μg / mL or about 1 μg / mL, 5 μg / mL or about 5 μg / mL, 5 μg / mL or about 5 μg / mL, 25 μg / mL or about 25 μg / mL, 5 μg / mL or about 5 μg / mL, 10 μg / mL or about 10 μg / mL, and 10 μg / mL or about 10 μg / mL, 25 μg / mL or about 25 μg / mL (inclusive). [This invention 1049] Any of the methods of claims 1008, 1009, 1017, 1018, and 1019 to 1027, wherein the checkpoint inhibitor inhibits the activity of an immune checkpoint selected from the group consisting of PD-1 / PD-L1, CTLA-4, OX40, LAG-3, TIM-3, and B7-H3. [The present invention 1050] 1049. The method of claim 1049, wherein said immune checkpoint is selected from PD-1 / PD-L1. [This invention 1051] 1050. The method of claim 1050, wherein said checkpoint inhibitor is an anti-PD-1 antibody, and optionally said antibody is selected from pembrolizumab, cemiplimab, nivolumab, or an antigen-binding fragment of any of the foregoing. [This invention 1052] 1052. The method of claim 1050 or claim 1051, wherein said checkpoint inhibitor is pembrolizumab. [This invention 1053] 1050. The method of claim 1050, wherein said checkpoint inhibitor is an anti-PDL1 antibody, and optionally said antibody is selected from avelumab, durvalumab, and atezolizumab, or an antigen-binding fragment of any of the foregoing. [This invention 1054] 1049. The method of claim 1049, wherein said immune checkpoint is OX40. [This invention 1055] 1054. The method of claim 1054, wherein said checkpoint inhibitor is an anti-OX40L antibody, and optionally, said antibody is oxelumab or an antigen-binding fragment thereof. [The present invention 1056] 1049. The method of claim 1049, wherein said immune checkpoint is CTLA-4. [This invention 1057] 1056. The method of claim 1056, wherein said checkpoint inhibitor is an anti-CTLA-4 antibody, and optionally, said antibody is ipilimumab or an antigen-binding fragment thereof. [This invention 1058] The checkpoint inhibitor is from 0.5 μg / mL or about 0.5 μg / mL to 25 μg / mL or about 25 μg / mL, 0.5 μg / mL or about 0.5 μg / mL to 10 μg / mL or about 10 μg / mL, 0.5 μg / mL or about 0.5 μg / mL to 5 μg / mL or about 5 μg / mL, 0.5 μg / mL or about 0.5 μg / mL to 1 μg / mL or about 1 μg / mL, 1 μg / mL or about 1 μg / mL to 25 μg / mL or about 25 μg / mL, 1 μg / mL or about 1 μg / mL to 10 μg / mL Any of methods 1008, 1009, 1017, 1018, 1019-1027, and 1049-1057, wherein the hydroxybenzoate is added at a concentration of from 1 μg / mL or about 1 μg / mL, 5 μg / mL or about 5 μg / mL, 5 μg / mL or about 5 μg / mL, 25 μg / mL or about 25 μg / mL, 5 μg / mL or about 5 μg / mL, 10 μg / mL or about 10 μg / mL, and 10 μg / mL or about 10 μg / mL to 25 μg / mL or about 25 μg / mL (inclusive). [This invention 1059] Any of the methods of claims 1001 to 1058, wherein the T cell adjuvant is added continuously during one or more stages of the culturing process, and the T cell adjuvant is replenished or replaced one or more times during one or more stages of the culturing process. [The present invention 1060] Any of the methods of claims 1002 to 1009, 1011 to 1019, and 1020 to 1059, wherein the T cell adjuvant is added continuously during incubation with the one or more recombinant cytokines, and the T cell adjuvant is replenished or replaced one or more times during incubation. [The present invention 1061] Any of the methods of inventions 1001 to 1058, wherein the T cell adjuvant is added temporarily during one or more stages of the culturing process, and the T cell adjuvant is added only once during one or more stages of the culturing process. [The present invention 1062] Any of the methods of claims 1002 to 1009, 1011 to 1019, and 1020 to 1058, and 1061, wherein the T cell adjuvant is added transiently during incubation with the one or more recombinant cytokines, and the T cell adjuvant is added only once during the incubation. [The present invention 1063] 10. The method of any of claims 1001 to 1062, wherein the first T cell stimulating agent comprises a recombinant cytokine selected from the group consisting of IL-10, IL-1a, IL-5, IL-7, IL-12, IL-23p40, IL-16, IL-17A, IL-15, IL-22, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL13, and IL-1b. [This invention 1064] 13. The method of any of claims 1001 to 1062, wherein the second T cell stimulator comprises a recombinant cytokine selected from the group consisting of IL-10, IL-1a, IL-5, IL-7, IL-12, IL-23p40, IL-16, IL-17A, IL-15, IL-22, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL13, and IL-1b. [This invention 1065] The method of any of claims 1002 to 1009, 1011 to 1019, and 1020 to 1064, wherein the recombinant cytokine is selected from one or more of IL-2, IL-15, IL-7, and IL-21. [The present invention 1066] The method of any of claims 1002 to 1009, 1011 to 1019, and 1020 to 1065, wherein the one or more recombinant cytokines include recombinant IL-2. [This invention 1067] 1. A method for producing tumor-reactive T cells, comprising: (1)(a) incubating a cell population comprising T cells from a biological sample obtained from a subject having a tumor with a first T cell stimulator comprising one or more recombinant cytokines selected from one or more of IL-7, IL-15, and IL-21 under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject, thereby generating a population containing T cells, including tumor-reactive T cells; (d) enriching the co-culture for a tumor-reactive T cell population that is reactive to the one or more peptides, thereby producing a T cell population enriched for tumor-reactive T cells, wherein the tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen; and (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator under conditions to stimulate expansion of T cells within the population. Culturing the T cells by a process comprising: (2) harvesting the expanded cells produced by the method; The method comprises: [The present invention 1068] 1068. The method of claim 1067, wherein said first T cell stimulator comprises one or more recombinant cytokines, including IL-7 and IL-15. [This invention 1069] The method of any of claims 1066 to 1068, wherein said first T cell stimulating agent further comprises recombinant IL-2. [The present invention 1070] The method of any of claims 1066 to 1069, wherein said first T cell stimulating agent does not comprise recombinant IL-2. [This invention 1071] 10. The method of any of claims 1066 to 1070, wherein the second T cell stimulator comprises one or more recombinant cytokines selected from the group consisting of IL-10, IL-1a, IL-5, IL-7, IL-12, IL-23p40, IL-16, IL-17A, IL-15, IL-22, IL-2, IL-4, IL-6, IL-8, IL-10, IL12p70, IL13, and IL-1b. [This invention 1072] 1072. The method of claim 1071, wherein said recombinant cytokine is selected from one or more of IL-2, IL-15, IL-7, and IL-21. [This invention 1073] 1073. The method of claim 1071 or 1072, wherein said one or more recombinant cytokines comprises recombinant IL-2. [This invention 1074] 10. The method of any one of claims 1002 to 1009, 1011 to 1019, and 1020 to 1073, wherein the concentration of each of said one or more recombinant cytokines is individually 100 IU / mL to 6000 IU / mL. [This invention 1075] 10. The method of any one of claims 1002 to 1009, 1011 to 1019, and 1020 to 1064, wherein the concentration of each of said one or more recombinant cytokines is, individually, 300 IU / mL to 6000 IU / mL, 300 IU / mL to 3000 IU / mL, or 300 IU / mL to 1000 IU / mL, and optionally the concentration of each of said one or more recombinant cytokines is 300 IU / mL or about 300 IU / mL, or 1000 IU / mL or about 1000 IU / mL. [This invention 1076] The method of any of claims 1001 to 1075, wherein the second T cell stimulator further comprises an anti-CD3 antibody, optionally OKT3, and optionally the concentration of the anti-CD3 antibody is 50 ng / mL or about 50 ng / mL. [This invention 1077] The method of any of claims 1001 to 1076, wherein said antigen-presenting cells are nucleated cells, such as dendritic cells, mononuclear phagocytes, B lymphocytes, endothelial cells, or thymic epithelium. [This invention 1078] The method of any one of claims 1001 to 1077, wherein the antigen-presenting cells are dendritic cells. [This invention 1079] The method of any of claims 1001 to 1078, wherein said antigen presenting cells are autologous to the subject or allogeneic to the subject. [The present invention 1080] An antigen-presenting cell, any of the methods of claims 1001 to 1079. [This invention 1081] The method of any of claims 1001 to 1080, wherein said T cells are autologous to said subject. [This invention 1082] The method of any of claims 1001 to 1081, wherein said one or more peptides comprise at least one neoepitope derived from a tumor-associated antigen from said subject. [This invention 1083] The co-culture (a) identifying somatic mutations associated with one or more tumor-associated antigens by exome sequencing of healthy and tumor tissues from a subject; (b) identifying at least one neoepitope of said one or more tumor-associated antigens; (c) isolating a population of autologous T cells from a biological sample from the subject; and (d) co-culturing the T cell population with antigen-presenting cells (APCs) that have been exposed to or contacted with one or more peptides comprising at least one neoepitope of the one or more tumor-associated antigens under conditions for presenting one or more of said peptides on the surface of the major histocompatibility complex (MHC), thereby generating a T cell population comprising tumor-reactive T cells that are reactive to one or more of said peptides. Any of the methods of inventions 1001 to 1082, which is carried out by a process comprising: [This invention 1084] The method of any one of claims 1001 to 1083, wherein the MHC molecule is a class I molecule. [This invention 1085] The method of any one of claims 1001 to 1083, wherein the MHC molecule is a class II molecule. [This invention 1086] The method of any of claims 1001 to 1083, wherein the MHC molecules are MHC class I and II. [This invention 1087] The method of any one of claims 1001 to 1086, wherein said T cells are CD4+ cells. [This invention 1088] The method of any one of claims 1001 to 1086, wherein the T cells are CD8+ cells. [This invention 1089] The method of any one of claims 1001 to 1086, wherein said T cells are CD4+ cells and CD8+ cells. [The present invention 1090] The method of any of claims 1001 to 1089, wherein said one or more peptides comprise an individual peptide or a peptide pool. [This invention 1091] 1090. The method of any of claims 1001 to 1090, wherein the one or more peptides are loaded onto antigen-presenting cells by transfection of an in vitro transcribed synthetic minigene construct encoding the one or more peptides, optionally flanked in tandem on either side by 12 amino acids from an endogenous protein, and the transcribed minigene construct generates the individual peptides. [This invention 1092] The method of any of claims 1001 to 1090, wherein said one or more peptides are loaded onto antigen-presenting cells by peptide pulsing, optionally by electroporation. [This invention 1093] 1092. The method of claim 1092, wherein said one or more peptides are each individually 5 to 30 amino acids in length, optionally 12 to 25 amino acids in length, optionally 25 amino acids in length or about 25 amino acids in length. [This invention 1094] the one or more peptides are a peptide pool, and the concentration of the peptides in the peptide pool for the peptide pulse is from 0.001 μg / mL or about 0.001 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL to 40 μg / mL or about 40 μg / mL, 0.1 μg / mL or about 0.1 μg / mL to 40 μg / mL or about 40 μg / mL, 1 μg / mL or about 1 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL or about 0.01 μg / mL to 10 μg / mL or about 10 μg / mL, or 1 μg / mL or about 1 μg / mL to 10 μg / mL or about 10 μg / mL; or The one or more peptides are individual peptides, and the concentration of the individual peptide for the peptide pulse is from 0.00001 μg / mL or about 0.00001 μg / mL to 1 μg / mL or about 1 μg / mL, 0.00001 μg / mL or about 0.00001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, 0.00001 μg / mL or about 0.00001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL, 0. 0.0001 μg / mL or about 0.0001 μg / mL to 1 μg / mL or about 1 μg / mL, 0.0001 μg / mL or about 0.0001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, 0.0001 μg / mL or about 0.0001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, or 0.0001 μg / mL or about 0.0001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL, The method of invention 1092 or invention 1093. [This invention 1095] 15. The method of any of claims 1092 to 1094, wherein the concentration of each individual peptide of said one or more peptides is, on average, from at or about 0.00001 μg / mL to at or about 0.01 μg / mL. [This invention 1096] 1095. The method of any of claims 1092 to 1094, wherein the concentration of each individual peptide of said one or more peptides is, on average, from 0.0001 μg / mL or about 0.0001 μg / mL to 0.001 μg / mL or about 0.001 μg / mL. [This invention 1097] Any of the methods of inventions 1001 to 1096, wherein the co-culture ratio of antigen-presenting cells and T cells is 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 5:1 to 1:1, 2.5:1 to 1:1, 1:20 to 1:1, 1:15 to 1:1, 1:10 to 1:1, 1:5 to 1:1, or 1:2.5 to 1:1. [This invention 1098] The method of any of claims 1001 to 1097, wherein the co-culture ratio of antigen-presenting cells to T cells is 1:1 or about 1:1. [This invention 1099] The method of any one of claims 1001 to 1098, wherein the co-culture lasts for 2 hours to 24 hours. [The present invention 1100] 1099. The method of any of claims 1001 to 1099, wherein said co-cultivation lasts for 6 hours or about 6 hours. [The present invention 1101] The method of any of claims 1001 to 1100, wherein said enriching for tumor-reactive T cells comprises selecting T cells that are surface positive for one or more T cell activation markers. [The present invention 1102] 1101. The method of claim 1101, wherein said one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD69, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, and LAG-3. [The present invention 1103] 1103. The method of any of claims 1001 to 1102, wherein said one or more T cell activation markers are selected from the group consisting of CD38, CD39, CD6, CD90, CD134, and CD137. [The present invention 1104] The method of any of claims 1001 to 1103, wherein said one or more T cell activation markers are CD134 and / or CD137. [This invention 1105] Any of the methods of claims 1001 to 1104, wherein said one or more T cell activation markers are selected from the group consisting of CD107, CD107a, CD39, CD103, CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, and CD256. [The present invention 1106] 1106. The method of any of claims 1001 to 1102 and 1105, wherein said one or more T cell activation markers are selected from the group consisting of CD107a, CD39, CD103, CD59, CD90, and CD38. [This invention 1107] 1106. The method of any of claims 1001 to 1102, 1105, and 1106, wherein the one or more T cell activation markers comprise at least two markers selected from CD107a and CD39, CD107a and CD103, CD107a and CD59, CD107a and CD90, CD107a and CD38, CD39 and CD103, CD39 and CD59, CD39 and CD90, CD39 and CD38, CD103 and CD59, CD103 and CD90, CD103 and CD38, CD59 and CD90, CD59 and CD38, and CD90 and CD38. [This invention 1108] 1108. The method of any of claims 1105 to 1107, wherein said one or more T cell activation markers further comprise CD137. [This invention 1109] 1108. The method of claim 1108, wherein said one or more T cell activation markers comprise at least two markers selected from CD107a and CD137, CD38 and CD137, CD103 and CD137, CD59 and CD137, CD90 and CD137, and CD38 and CD137. [The present invention 1110] 1109. The method of any of claims 1103 to 1109, wherein said one or more T cell activation markers further comprise at least one marker selected from the group consisting of PD-1, TIM-3, and LAG-3. [The present invention 1111] 11. The method of any of claims 1101 to 1110, wherein selecting T cells surface positive for said one or more T cell activation markers is by flow cytometry, optionally by automated high throughput flow cytometry, optionally by an FX500 cell sorter or a Miltenyi Tyto cell sorter. [The present invention 1112] 1111. The method of claim 1111, wherein one run, two runs, three runs, or four runs by flow cytometry are performed to enrich said tumor-reactive T cells from said sample. [The present invention 1113] The method of any of claims 1001 to 1112, wherein one or more of the method steps are carried out in a closed system. [This invention 1114] The method of any of claims 1001 to 1113, wherein the incubation with said first T cell stimulating agent is for 7 to 21 days, optionally 7 to 14 days. [This invention 1115] The method of any of claims 1001 to 1114, wherein the incubation with the first T cell stimulating substance is carried out in a closed system. [The present invention 1116] The method of any of claims 1001 to 1115, wherein the incubation with the first T cell stimulating substance is carried out in a gas-permeable culture vessel. [This invention 1117] The method of any of claims 1001 to 1116, wherein the incubation with the first T cell stimulating substance is carried out using a bioreactor. [This invention 1118] The method of any of claims 1001 to 1117, wherein the incubation with said second T cell stimulator is for 7 to 21 days, optionally for 7 to 14 days. [This invention 1119] The method of any of claims 1001 to 1118, wherein the incubation with the second T cell stimulating substance is carried out in a closed system. [The present invention 1120] The method of any of claims 1001 to 1119, wherein the incubation with the second T cell stimulating substance is carried out in a gas-permeable culture vessel. [This invention 1121] The method of any of claims 1001 to 1119, wherein the incubation with the second T cell stimulating substance is carried out using a bioreactor. [This invention 1122] The method of any of claims 1001 to 1121, wherein the harvesting step is carried out within 30 days after the start of culturing and / or enriching the T cells, including tumor-reactive cells. [This invention 1123] 13. The method of any of claims 1001 to 1122, wherein the cells are harvested up to 30 days after initiation of said culturing, and optionally 7 to 30 days, 7 to 20 days, 7 to 14 days, 7 to 10 days, 10 to 20 days, 10 to 14 days, or 14 to 20 days after initiation of said culturing. [This invention 1124] The method of any one of claims 1001 to 1123, wherein the subject exhibits cancer. [Invention 1125] The method of any of claims 1001 to 1124, wherein a composition comprising the expanded cells produced by said method is used to treat cancer in said subject. [The present invention 1126] The method of any one of claims 1001 to 1125, wherein the tumor is an epithelial cancer tumor. [This invention 1127] The method of any one of claims 1001 to 1126, wherein said tumor is melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, gastric cancer, or uterine cancer. [This invention 1128] Any of the methods of claims 1001 to 1127, wherein the tumor is a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer tumor, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer. [This invention 1129] The method of any of claims 1001 to 1128, wherein said biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample. [The present invention 1130] 1129. The method of claim 1129, wherein said biological sample is a peripheral blood sample, said peripheral blood sample being collected by blood collection or apheresis, and optionally said apheresis being leukapheresis. [This invention 1131] 1129. The method of claim 1129, wherein said biological sample is a lymph node sample or a tumor sample, and said sample is collected by needle biopsy, optionally core needle biopsy, or fine needle aspiration. [This invention 1132] The method of any of claims 1001 to 1131, wherein said cell population comprising T cells comprises tumor-infiltrating lymphocytes, lymphocytes, or peripheral blood mononuclear cells. [This invention 1133] The method of any of claims 1001 to 1132, wherein said biological sample is a tumor, and said cell population containing T cells comprises tumor-infiltrating lymphocytes. [This invention 1134] The method of any of claims 1001 to 1133, wherein said biological sample is a resected tumor, and said cell population comprising T cells is one or more tumor fragments derived from the resected tumor. [This invention 1135] 1135. The method of claim 1134, wherein said one or more tumor fragments are seeded for incubation with said first T cell stimulator at about 1 tumor fragment / 2 cm2. [This invention 1136] The method of any one of claims 1001 to 1134, wherein the tumor is melanoma. [This invention 1137] Any of the methods of claims 1001 to 1134, wherein the biological sample is a resected tumor and the cell population comprising T cells is processed as a single cell suspension by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor. [This invention 1138] Any of the methods of claims 1001 to 1134, wherein the biological sample is a resected tumor and the cell population comprising T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from the resected tumor. [This invention 1139] The method of claim 1137 or claim 1138, wherein said enzymatic digestion is by incubation with collagenase, optionally collagenase IV or collagenase I / II. [The present invention 1140] The cell population containing T cells is about 5×10 5 From 2 x 10 6 pieces or approximately 2 x 10 6 total cells / 2cm 2 The method of any of claims 1137 to 1139, wherein the cells are seeded for incubation with said first T cell stimulator. [This invention 1141] The method of any of claims 1001 to 1135 and 1137 to 1140, wherein said tumor is colorectal cancer (CRC). [This invention 1142] Any of the methods of inventions 1001 to 1141, which results in a fold increase in T cells or a fold increase in tumor-reactive T cells of at least 2-fold or at least about 2-fold, at least 5-fold or at least about 5-fold, at least 10-fold or at least about 10-fold, at least 25-fold or at least about 25-fold, at least 50-fold or at least about 50-fold, at least 100-fold or at least about 100-fold, at least 250-fold or at least about 250-fold, at least 500-fold or at least about 500-fold, at least 1000-fold or at least about 1000-fold, or more. [This invention 1143] 13. The method of any of claims 1001 to 1142, wherein said composition of expanded cells is capable of producing IFNγ, following antigen-specific stimulation, at a concentration of greater than or greater than about 30 pg / mL, optionally greater than or greater than about 60 pg / mL. [This invention 1144] The method of any of claims 1001 to 1143, comprising the step of formulating the collected cells using a cryoprotectant. [Invention 1145] Expanded T cells enriched for tumor-reactive T cells produced by any of the methods of the present inventions 1001 to 1144 A composition comprising: [Invention 1146] A composition of the present invention 1145 comprising a pharmaceutically acceptable excipient. [This invention 1147] The composition of invention 1145 or invention 1146, wherein said T cells are CD3+ T cells or comprise CD4+ T cells and / or CD8+ T cells. [Invention 1148] Any of the compositions of the present inventions 1145 to 1147, wherein the T cells comprise CD4+ T cells and CD8+ T cells, and the ratio of CD8+ T cells to CD4+ T cells is from 1:100 or about 1:100 to 100:1 or about 100:1, 1:50 or about 1:50 to 50:1 or about 50:1, 1:25 or about 1:25 to 25:1 or about 25:1, 1:10 or about 1:10 to 10:1 or about 10:1, 1:5 or about 1:5 to 5:1 or about 5:1, or 1:2.5 or about 1:2.5 to 2.5:1 or about 2.5:1. [This invention 1149] The number of tumor-reactive T cells in the composition, or the total number of T cells surface-positive for the T cell activation marker, or the number of viable cells thereof, is 0.5×10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 0.5×10 8 From 12 x 10 9 pieces or approximately 12 x 109 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 3.5 x 10 8 or approximately 3.5 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 pieces, 1×10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 1×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 30 x 109 pieces or approximately 30 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 60×10 8 pieces or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 pieces or approximately 60 x 10 9 1149. The composition of any one of 1145 to 1148, wherein the number of the compounds is 1149 (including both end values). [This invention 1150] Any of the compositions of claims 1145 to 1149, which comprises a cryoprotectant. [This invention 1151] Any of the compositions of inventions 1145 to 1150, which is sterile. [This invention 1152] A method for treating a subject having cancer, comprising the step of administering a therapeutic dose of any of the compositions of present inventions 1145 to 1151 to a subject having a tumor. [This invention 1153] The therapeutically effective dose is 1×10 9 ~10×10 9 The method of claim 1152, wherein the T cells are T cells. [This invention 1154] The method of claim 1152 or claim 1153, wherein said cells of said composition to be administered are autologous to said subject. [Invention 1155] The method of any one of claims 1152 to 1154, wherein the cancer is epithelial cancer. [Invention 1156] The method of any one of claims 1152 to 1154, wherein said cancer is melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer. [This invention 1157] Any of the methods of claims 1152 to 1154, wherein the cancer is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer. [Brief explanation of the drawings]

[0144] [Figure 1]Figure 1A shows a schematic diagram of an exemplary process for manufacturing a T cell therapy composition according to the provided methods. In the exemplary process, a tumor sample is obtained from a patient for identification and generation of peptides for use in co-culture with autologous T cells obtained from the same subject. In some cases, a T cell population obtained from the patient, containing, for example, tumor-infiltrating lymphocytes (TILs) or peripheral blood lymphocytes (PBLs), is stimulated under conditions for cell expansion before co-culture with antigen-presenting cells that have been contacted or exposed to peptide neoepitopes for presentation on the major histocompatibility complex. After co-culture under conditions in which the antigen-presenting cells present the peptide in the context of the major histocompatibility complex, tumor-reactive T cells, or T cells surface-positive for one or more T cell activation markers associated with tumor-reactive T cells (e.g., CD70a), can be selected and cultured under conditions for expansion according to the provided methods, such as incubation with a T cell stimulator (e.g., anti-CD3 / anti-CD28). According to the provided methods, steps may include incubation with a T cell adjuvant that is a costimulatory agonist (e.g., an OX40 or 4-1BB agonist) or an apoptosis inhibitor (e.g., a Fas / Fas ligand inhibitor or a caspase inhibitor). The culturing step may be performed in the presence of one or more recombinant cytokines (e.g., IL-2) to support cell growth and expansion. This process may be performed in the presence of a serum-free medium containing nutrients. One or more, or all, of the steps may be performed in a closed system, e.g., without exposing the cells to the environment. Once a therapeutic dose or threshold number of cells is reached, the cells are harvested and formulated, optionally concentrated or cryopreserved, and available for administration to a subject, e.g., by infusion. Figure 1B shows a schematic diagram of an exemplary process for manufacturing a T cell therapy composition according to the provided methods. In the exemplary process, a biological sample containing T cells is used as the cell source for the method. The biological sample may include tumor-infiltrating lymphocytes, peripheral blood mononuclear cells (e.g., apheresis), or lymph-derived lymphocytes.Tumor-reactive T cells, or T cells surface-positive for one or more T cell activation markers associated with tumor-reactive T cells (e.g., CD70a), can be directly selected from a sample and cultured under conditions for expansion according to the provided methods, including incubation with a T cell adjuvant that is a costimulatory agonist (e.g., OX40 or 4-1BB agonist) or an apoptosis inhibitor (e.g., a Fas / Fas ligand inhibitor or a caspase inhibitor), and incubation with a T cell stimulator (e.g., anti-CD3 / anti-CD28). The culturing step can be performed in the presence of one or more recombinant cytokines (e.g., IL-2) to support cell growth and expansion. This process can be performed in the presence of serum-free medium containing nutrients. One or more, or all, of the steps can be performed in a closed system, e.g., without exposing the cells to the environment. Once a therapeutic dose or threshold number of cells is reached, the cells can be harvested, formulated, optionally concentrated or cryopreserved, and used for administration to a subject, e.g., by infusion. Figure 1C shows the complete process flow chart for generating patient-specific tumor-derived infiltrating T cell populations. [Figure 2] Figure 2A shows exemplary kinetics and T cell neoantigen reactivity in a typical TIL expansion process, which includes a bulk expansion of T cells with a first initial expansion and a second rapid expansion, where reactivity remains low throughout the process, including in the final product. Figure 2B further shows exemplary kinetics of a TIL expansion process, which includes a first initial expansion, followed by enrichment of tumor-reactive T cells by co-culture with neoantigen peptide-presenting antigen-presenting cells, selection of tumor-reactive cells for T cell activation (upregulation) markers, and a second expansion of the enriched reactive cells. [Figure 3]Figure 3A shows the generation of viable whole population 1 cells from patient-derived CRC tumor tissue using fragment culture and enzymatic and non-enzymatic homogenization. Both enzymatic and non-enzymatic digestion yielded more whole cells than cultures from fragments. The viability of these cells is shown in Figure 3B. The viability of enzymatically digested cultures generated from fragments was higher than that obtained using non-enzymatic homogenization. [Figure 4] Figure 4A shows the generation of Population 1 cells from patient-derived melanoma tumor tissue using fragment culture or homogenization with or without enzymes. Fragment cultures yielded more total cells than cultures initiated from single-cell suspensions. The viability of these cells is shown in Figure 4B. Populations generated from fragments exhibited higher viability than cells from single-cell suspensions. [Figure 5] Figure 5A shows the growth curves (Figure 5B) and fold expansion (Figure 5C) of Population 2 cells derived from primary CRC tumors in either conventional 6-well culture plates or 24-well gas-permeable culture plates. Figure 5 also shows the total cell number (Figure 5C) and fold expansion (Figure 5D) of Population 2 cells derived from primary CRC tumors compared by cell extraction method, either fragment culture or single-cell suspension culture. [Figure 6] Growth curves (FIG. 6A) and fold expansion (FIG. 6B) of Population 2 cells derived from primary melanoma tumors in either 6-well culture plates or 24-well gas-permeable culture plates are shown. [Figure 7] The total cell number (FIG. 7A) and fold expansion (FIG. 7B) of Population 2 cells derived from primary CRC tumors using serum-free OpTmizer medium or RPMI medium supplemented with 5% human serum are shown. [Figure 8] Similarly, Figure 8 shows the total cell number (Figure 8A) and fold expansion (Figure 8B) of Population 2 cells derived from primary melanoma tumors using serum-free OpTmizer medium or RPMI medium supplemented with 5% human serum. [Figure 9]Figure 9A shows the total cell number (Figure 9A) and fold expansion (Figure 9B) of Population 2 cells derived from CRC tumors and cultured in medium supplemented with either low (300 IU / mL) or high (600 IU / mL) concentrations of recombinant human IL-2. These data are similarly shown for cells derived from melanoma tumors. [Figure 10] Figure 10A-B. High concentrations of IL-2 were not observed to be necessary for cell expansion. [Figure 11] Figure 11A shows the total cell counts of population 2, and Figure 11B shows that similar fold expansions were observed from melanoma-derived cell cultures that were unstimulated or stimulated with the anti-CD3 monoclonal antibody OKT3. [Figure 12] Figures 12A-C show the upregulation rates of activation markers on CD8+ T cells, CD38 and CD39 (Figure 12A), CD134 and CD137 (Figure 12B), and CD69 and CD90 (Figure 12C), from 0 to 48 hours after activation with OKT3. [Figure 13] Figures 13A-C show the upregulation rates of activation markers on CD4+ T cells, CD38 and CD39 (Figure 13A), CD134 and CD137 (Figure 13B), and CD69 and CD90 (Figure 13C), from 0 to 48 hours after activation with OKT3. [Figure 14] 1 shows the expression of selected exemplary markers in single cell suspension cultures generated from day 0 CRC tumors. [Figure 15]Figures 15A-E show CD3+ cell purity as a percentage of population 1 cells. Figure 15A shows the purity of cells derived from day 0 SCS from CRC tumors after homogenization without enzyme, homogenization with 1 mg / ml (low) enzyme, and homogenization with 5 mg / ml (high) enzyme. These data are similarly shown in Figure 15B for melanoma-derived cultures. Figure 15C shows the purity of CD3+ population 1 cells on day 0 (baseline SCS) and day 6 from fragments cultured with or without OKT3 stimulation. Figure 15D shows the relative purity of CD3+ cells from CRC donors on day 11 using fragments cultured in medium supplemented with either 6000 IU / mL (high) or 300 IU / mL (low) recombinant IL-2. Figure 15E shows population 1 cells (day 9) from fragments cultured in either serum-free OpTmizer medium or RPMI containing OKT3 stimulation and / or either high or low concentrations of IL-2. These observations support the idea that SCS derived from tumor biopsies of CRC patients may provide greater numbers of T cells for expansion than cells obtained from culture of tumor fragments. [Figure 16] 1 shows the purity of CD3+ Population 1 cells from melanoma patients as fragment cultures from day 9 using serum-containing RPMI medium or serum-free OpTmizer at high and low IL-2 concentrations. [Figure 17] Figure 17A shows the generation of Population 3 cells after co-culture with dendritic cells loaded with peptides at concentrations ranging from 0.1 ng / mL to 20 ng / mL. Figure 17B shows the fold increase in the same experiment from T cells co-cultured with non-loaded dendritic cells (Figure 17B). [Figure 18] Figure 18A compares stimulation with one or two peptides, reported as % 41BB / OX40 expression, and Figure 18B shows stimulation with one or two peptides, reported as fold increase from resting T cells. [Figure 19]Figure 19A compares two T cell to dendritic cell ratios of 1:1 and 1:2, reported as % 41BB / OX40 expression. Figure 19B compares two T cell to dendritic cell ratios of 1:1 and 1:2, reported as fold increase from resting T cells. [Figure 20] Figure 20A shows the neoantigen-reactive TCR rates before and after co-culture with autologous neoantigen peptides and sorting of T cells from peripheral blood of three healthy donors. Figure 20B shows the average class I reactivity of CD8+ cells before and after co-culture and sorting. [Figure 21] Figures 21A and 21B show the recovery from cell sorting using the Sony FX500, both as total cell input and output, for two independent runs (Figure 21A) and recovery (Figure 21B). [Figure 22] Figure 1 shows the purity and gating of the CD4+ population from cell sorting using a Sony FX500. The results demonstrate the selection of cells positive for up-regulation markers and high recovery of cells after sorting. [Figure 23] Figures 23A-C show the expansion of tumor-infiltrating T lymphocytes after sorting. Figure 23A shows the total cell count, and Figure 23B shows the fold expansion of population 5 cells from population 4 cells after co-culture with or without dendritic cells loaded with wild-type peptide, tumor-associated peptide, or no peptide. The predicted cell numbers after expansion of population 4 cells to population 5 cells at various cell recovery numbers after sorting are shown in Figure 23C. [Figure 24]Figure 24A shows measured IFN-γ secretion in bulk coculture positively selected (selected) populations or negatively selected (unselected) populations from bulk coculture cells (enriched) following stimulation with mutant (mutated) or normal wild-type (WT) peptides from ovarian cancer patients. Figure 24B shows enrichment of neoantigen-specific populations of tumor-reactive specific cells in positively and negatively selected populations compared to bulk unsorted T cells. Figure 24C shows the number of TCR clonotypes present in unselected and selected populations, indicating high incoming TCR diversity in unsorted T cell populations and enrichment of unique TCR clones in selected populations. Figure 24D shows pre- and post-sort cell populations from sample A observed to contain CD4+ and CD8+ cells, demonstrating the presence of class I and class II reactive cells in the enriched population. [Figure 25] Figure 25A shows measured IFN-γ secretion within a bulk coculture positively selected (selected) population or a negatively selected (unselected) population from bulk coculture cells (enriched) after stimulation with anti-CD3 (OKT3) obtained from a colorectal cancer patient. Figure 25B shows enrichment of neoantigen-specific populations of tumor-reactive specific cells in positively and negatively selected T cells compared to bulk unselected T cells. Figure 25C shows the TCR clonality profiles present in unselected and selected populations. Figure 25D shows pre- and post-sort cell populations observed to contain CD4+ and CD8+ cells, indicating that class I and class II reactive cells are present in the enriched population. [Figure 26]Figure 26A shows the enrichment of neoantigen-specific populations of tumor-reactive specific cells in bulk coculture positively sorted (selected) populations or negatively sorted (unselected) populations from bulk coculture cells (enriched) by expression of CD137 and / or CD134. Figure 26B shows the TCR clonality profiles present in unselected and selected populations. Figure 26C shows pre-sort (bulk) and post-sort cell populations observed to contain both CD4+ class I-reactive and CD8+ class II-reactive cells. [Figure 27] Figure 27A-C shows the total viable CD3+ cell counts of cells grown in the presence of multiple T cell adjuvants with supplemental OKT3 stimulation. Results shown are for the following adjuvants: 10 μg / mL of tabolixizumab, oxelumab, ipilimumab, tocilizumab, urelumab, pembrolizumab, varlilumab, anti-GITR MK-1248, anti-human FasL; 25 μM for the Z-VAD-FMK pan-caspase inhibitor; 250 nM for the HSP inhibitor NVP-HSP990; and 1000 IU / mL for cytokines (IL-7, IL-15, or IL-21). [Figure 28] Figure 28A-C shows the total viable CD3+ cell counts of cells grown in the presence of multiple T cell adjuvants in the absence of supplemental OKT3 stimulation. Results shown are for the following adjuvants: 10 μg / mL of tabolixizumab, oxelumab, ipilimumab, tocilizumab, urelumab, pembrolizumab, varlilumab, anti-GITR MK-1248, anti-human FasL; 25 μM for the Z-VAD-FMK pan-caspase inhibitor; 250 nM for the HSP inhibitor NVP-HSP990; and 1000 IU / mL for cytokines (IL-7, IL-15, or IL-21). [Figure 29] Dose-response curves for IL-7 (Figure 29A) and IL-15 (Figure 29B) are shown. [Figure 30]The total cell number and cell viability of cells derived from each of the three healthy donors and grown under the experimental conditions described are shown in Figure 30A-B. Cells grown in the presence of continuous caspase inhibition were observed to exhibit superior proliferation despite inherent donor variability. [Figure 31] The total cell number and cell viability of cells derived from each of the three healthy donors and grown under the experimental conditions described are shown in Figures 31A-B. Cells grown in the presence of continuous caspase inhibition were observed to exhibit superior proliferation despite inherent donor variability. [Figure 32] The total cell numbers and cell viability of cells derived from each of the three healthy donors and grown under the experimental conditions described are shown in Figures 32A-B. Cells grown in the presence of continuous caspase inhibition were observed to exhibit superior proliferation despite inherent donor variability. [Figure 33] The cell survival rates of two donors after single activation with anti-CD3 / anti-CD28 (transient activation) treatment group are shown in Figures 33A and 33B. [Figure 34] The total cell counts for the same treatments as in Figures 33A-B are shown in Figures 34A-B. [Figure 35] The cell survival rates of two donors after continuous activation in the anti-CD3 / anti-CD28 treatment group are shown in Figures 35A and 35B. [Figure 36] The total cell counts for the same treatments as in Figures 35A-B are shown in Figures 36A-B. [Figure 37] Figures 37A-C show the fold expansion (Figure 37A), total viable cells (Figure 37B), and viability (Figure 37C) of both SCS and tumor fragment-derived cultures grown in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK. [Figure 38] T cell phenotype is shown for CD3+ (Figures 38A-D) cells grown in the presence of various concentrations of ipilimumab (anti-CTLA4), pembrolizumab (anti-PD1), taborixizumab (anti-TNFRSF4), urelumab (anti-CD137), and varlilumab (anti-CD27). [Figure 39]T cell phenotype is shown for CD4+ (Figure 39A-D) cells grown in the presence of various concentrations of ipilimumab (anti-CTLA4), pembrolizumab (anti-PD1), taborixizumab (anti-TNFRSF4), urelumab (anti-CD137), and varlilumab (anti-CD27). [Figure 40] T cell phenotype is shown for CD8+ (Figure 40A-D) cells grown in the presence of various concentrations of ipilimumab (anti-CTLA4), pembrolizumab (anti-PD1), taborixizumab (anti-TNFRSF4), urelumab (anti-CD137), and varlilumab (anti-CD27). DETAILED DESCRIPTION OF THE INVENTION

[0145] Detailed Description Provided herein are methods for producing T cells that express cell surface receptors that recognize peptides on the surface of target cells, such as tumors. The T cells can be tumor-reactive T cells that recognize tumor-associated antigens, e.g., neoantigens. The methods include culturing T cells ex vivo, where the T cells are isolated or obtained from a biological sample as a cellular source of the T cells. In some cases, the cellular source includes peripheral blood lymphocytes, lymph node-derived lymphocytes, or tumor-infiltrating lymphocytes. The cell culturing methods include cell growth and expansion methods, particularly including enrichment for the proliferation and expansion of tumor-reactive T cells, e.g., by selection of such cells or based on T cell activation markers associated with such cells. The provided methods also utilize specific T cell adjuvants in the ex vivo production of T cell therapy. In some embodiments, the T cell adjuvant is a costimulatory agonist. In some embodiments, the T cell adjuvant is an agent that inhibits apoptosis or apoptotic pathways in cells (hereinafter, "apoptosis inhibitors"). In some embodiments, the T cell adjuvant is an immune checkpoint modulator. In some embodiments, the T cell adjuvant is an agent that inhibits heat shock proteins or heat shock protein activity intracellularly. T cell adjuvants can be added to the production of T cells to enhance their functionality ex vivo and for use in in vivo therapeutic methods. In certain embodiments, such methods can enrich for the expansion of reactive T cells compared to non-responsive T cells and promote their survival and proliferation in ex vivo culture. It is believed that the provided methods can increase the expansion to a therapeutic dose to a much greater extent than existing methods and / or enhance the functionality of T cell therapy for therapeutic effect. The provided methods can be used to support the expansion and survival of donor cells ex vivo, for example, in connection with methods for producing T cell therapy for re-delivery to a patient donor or another patient.

[0146] The provided method relates to the generation of T cell therapies reactive against tumor-associated antigens, such as neoantigens. Cancer cells accumulate many different DNA mutations as part of the tumorigenesis process. These mutations can cause amino acid changes in protein-coding regions. For the mutations to be recognized by the immune system, the protein must be processed intracellularly and presented as a mutant peptide presented on the surface by the major histocompatibility complex (MHC). Peptide neoantigens (also referred to herein as neoepitopes or peptide neoepitopes) are mutant peptides presented by the MHC complex that can be recognized by T cells via TCR binding. For the immune system to recognize the mutation, the mutation must be expressed on the surface of the cancer cell via the MHC complex, and the T cell must have a TCR that recognizes the mutant peptide. These neoantigens can be presented by MHC class I and MHC class II and are recognized by CD8+ T cells and CD4+ T cells, respectively.

[0147] In certain embodiments of the provided methods, the T cell population is or includes reactive T cells that express a cell surface receptor, such as a T cell receptor (TCR), that can recognize peptide antigens on the surface of target cells. Specifically, for an antigen to be recognized by the immune system, the protein must be processed intracellularly into peptide fragments that are presented on the surface by major histocompatibility complex (MHC). TCRs have two protein chains designed to bind to specific peptides presented by major histocompatibility complex (MHC) proteins on the surface of specific cells. Because TCRs recognize peptides in the context of MHC molecules expressed on the surface of target cells, TCRs have the potential to recognize antigens not only presented directly on the surface of target cells, e.g., cancer cells, but also presented by antigen-presenting cells in, for example, tumor microenvironments, inflammatory and infectious microenvironments, and secondary lymphoid organs. Reactive T cells expressing such cell surface receptors can be used to target and kill any target cells, including, but not limited to, infected, damaged, or dysfunctional cells. Examples of such target cells may include cancer cells, virus-infected cells, bacteria-infected cells, dysfunctionally activated inflammatory cells (e.g., inflammatory endothelial cells), and cells involved in dysfunctional immune responses (e.g., cells involved in autoimmune diseases).

[0148] In some embodiments, a "T cell receptor" or "TCR" is a molecule that comprises a variable α chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively), or an antigen-binding portion thereof, and is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. TCRs, which typically exist in the αβ and γδ forms, are generally structurally similar, although the T cells that express them may have different anatomical locations or functions. TCRs can be found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0149] In some aspects, reactive T cells are tumor-reactive T cells that recognize cancer antigens. Cancer cells accumulate many different DNA mutations as part of the tumorigenesis process. These mutations can cause amino acid changes in protein-coding regions. Neoantigens are mutant peptides encoded by tumor-specific mutant genes and presented by the MHC complex, which can be recognized by T cells via TCR binding. For the immune system to recognize the mutations, neoantigens are expressed on the surface of cancer cells via the MHC complex for recognition by T cells with TCRs that recognize the mutant peptides. These neoantigens can be presented by MHC class I and MHC class II and are recognized by CD8+ T cells and CD4+ T cells, respectively. The majority of neoantigens arise from passenger mutations, which means they do not confer any growth advantage to cancer cells. A relatively small number of mutations actively promote tumor growth; these are known as driver mutations. Passenger mutations are likely to give rise to neoantigens that are unique to each patient and may be present in every subset of cancer cells. Driver mutations give rise to neo-antigens that are likely to be present and potentially shared in every tumor cell of an individual. In some embodiments of the provided methods, the T cell population contains tumor-reactive T cells that can recognize neo-antigens that contain passenger and / or driver mutations.

[0150] In certain aspects, the provided methods can be used for ex vivo generation of T cell therapy, including ex vivo expansion of autologous tumor-reactive T cells. In some aspects, neoantigens are ideal targets for immunotherapy because they are disease-specific targets. For example, such antigens are not commonly present in the body before cancer develops, are truly cancer-specific, are not expressed on normal cells, and are not subject to off-target immunotoxicity. Therefore, a patient's unique repertoire of neoantigens can elicit a potent immune response specific to cancer cells while avoiding normal cells. This is an advantage over other cell therapy targets that may not be disease-specific, as even low levels of target antigens on normal cells can result in severe, fatal autoimmune toxicity in the context of engineered therapies targeting common antigens. For example, in melanoma patients, an anti-MAGE-A3 TCR program was discontinued due to trial-related deaths caused by cross-reactivity with a similar target, MAGE-A12, which is expressed at low levels in the brain. A key challenge in cancer immunotherapy is the identification of cancer targets.

[0151] Recent clinical trials have demonstrated that T cells isolated from surgically resected tumors, bearing TCRs that recognize neoantigens, can expand the population of these neoantigen-reactive TILs, and reinfuse them into patients, potentially resulting in dramatic clinical benefit. This personalized therapy has resulted in remarkable clinical responses in selected patients with common epithelial tumors.

[0152] Existing methods for obtaining and generating tumor-reactive T cells are not entirely satisfactory. For example, directly isolating tumor-reactive T cells from a subject without expanding them is not feasible due to the inability to obtain therapeutically effective amounts of such cells. Alternatively, these cells can be identified by ex vivo co-culture of autologous bulk T cells in the presence of autologous antigen-presenting cells. In such methods, autologous antigen-presenting cells are exposed to or present source or potential tumor peptides to identify TCRs reactive to neoantigen mutations. While existing methods can generate reactive T cells, the procedures are often lengthy, require single-cell co-culture using droplet technology, and / or involve methods outside of GMP-controlled environments, posing safety risks related to endotoxins, mycoplasma, and sterility. Attempts have also been made to identify TCRs specific to desired neoantigens for recombinant engineering into T cells for use in adoptive cell therapy methods. However, such approaches only generate a single TCR against a particular neoantigen, thereby lacking the diversity to recognize a broader repertoire of multiple tumor-specific mutations. Other methods include bulk expansion of T cells from a tumor source, which carries the risk of expanding T cells that are not reactive to tumor antigens and / or may contain large numbers of bystander cells that may exhibit inhibitory activity. For example, tumor regulatory T cells (Tregs) are CD4+ T cells specialized in suppressing immune responses and may limit the reactivity of T cell products. + These additional approaches to expand tumor-reactive T cells ex vivo are not selective, and as a result, non-reactive T cells in the culture may be preferentially expanded over reactive T cells, resulting in an end product that lacks satisfactory reactivity and / or has insufficient numbers of tumor-reactive T cells. Methods for generating tumor-reactive T cells for therapy are needed.

[0153]

[0003] Provided embodiments relate to improved methods for identifying and expanding T cells ex vivo, including tumor-reactive T cells for use in T cell therapy. In some embodiments, the provided methods improve or increase the proliferation and survival of T cells, such as tumor-reactive T cells, outside the body. In certain embodiments, the methods enrich for the expansion of reactive T cells compared to non-reactive T cells, promoting their survival and proliferation in ex vivo culture. In some embodiments, the resulting methods can be performed in a closed system. In some embodiments, the methods are performed in an automated or partially automated manner.

[0154] In provided embodiments, the method comprises ex vivo incubation of cells enriched for a T cell population with a costimulatory agonist under conditions that stimulate or activate a costimulatory receptor expressed by one or more of the T cells in the sample. In certain embodiments, the costimulatory agonist is a 4-1BB agonist. In other particular embodiments, the costimulatory agonist is an OX40 agonist. One or more recombinant cytokines from recombinant IL-2, recombinant IL-7, recombinant IL-15, and / or recombinant IL-21 can also be included in the incubation to initially expand T cells within the cell population from the subject. In some embodiments, following incubation with the costimulatory agonist, or simultaneously with incubation with the costimulatory agonist, the T cell population is contacted with a T cell stimulator, e.g., recombinant IL-2, alone or in combination with one or more other recombinant cytokines (e.g., IL-7, IL-15, and / or IL-21), under conditions that induce or mediate proliferation of T cells within the population. In some embodiments, following incubation with the costimulatory agent, or concurrently with incubation with the costimulatory agent, the T cell population is contacted with a T cell stimulatory agent, e.g., an anti-CD3 antibody (e.g., OKT3) or an anti-CD3 / anti-CD28 stimulatory agent, e.g., anti-CD3 / anti-CD28 beads, under conditions that induce or mediate proliferation of T cells within the population. In some such aspects, a costimulatory agonist, such as a 4-1BB agonist or an OX40 agonist, provides an initial stimulus to enhance or promote the proliferative capacity and / or functional activity of T cells within the population.

[0155] In provided embodiments, the method comprises ex vivo incubation of cells enriched for a T cell population with an apoptosis inhibitor under conditions that reduce or prevent apoptosis of T cells in the sample. In certain embodiments, the apoptosis inhibitor is an inhibitor of the Fas / Fas ligand axis or an inhibitor of a caspase, both of which are involved in inducing apoptosis, particularly in activated T cells. In certain embodiments, the apoptosis inhibitor is an inhibitor of one or more caspases (also referred to as caspase inhibitors). As demonstrated herein, caspase inhibitors are found herein to significantly improve the expansion capacity of tumor-reactive T cells, particularly from patient tumors, or their proliferative capacity when the cells are activated under conditions that may be present in the tumor microenvironment. One or more recombinant cytokines, including recombinant IL-2, recombinant IL-7, recombinant IL-15, and / or recombinant IL-21, can also be included in the incubation to initially expand T cells within the cell population from the subject. In some embodiments, following incubation with the apoptosis inhibitor, or simultaneously with incubation with the apoptosis inhibitor, the T cell population is contacted with a T cell stimulant, e.g., recombinant IL-2, alone or in combination with one or more other recombinant cytokines (e.g., IL-7, IL-15, and / or IL-21), under conditions that induce or mediate proliferation of T cells within the population. In some embodiments, following incubation with the apoptosis inhibitor, or simultaneously with incubation with the apoptosis inhibitor, the T cell population is contacted with a T cell stimulant, e.g., an anti-CD3 antibody (e.g., OKT3), or an anti-CD3 / anti-CD28 stimulant, e.g., anti-CD3 / anti-CD28 beads, under conditions that induce or mediate proliferation of T cells within the population. In some such aspects, the apoptosis inhibitor protects T cells from apoptosis, thereby restoring the potential for T cells within the population to proliferate and expand.

[0156] In certain embodiments, the T cell adjuvant, e.g., costimulatory agonist, apoptosis inhibitor, immune checkpoint regulator, and / or heat shock protein inhibitor, is a soluble protein, e.g., a protein that is not bound or attached to a solid surface (e.g., a bead or other solid support). The T cell adjuvant may comprise a small molecule, peptide, or protein. Among such T cell adjuvants are soluble ligands, antibodies or antigen-binding fragments, or other binding agents. In some embodiments, the costimulatory agonist may comprise a molecule that specifically binds to a costimulatory molecule, such as 4-1BB or OX40, to induce or stimulate a costimulatory signal within the cell. In some embodiments, the apoptosis inhibitor may comprise a molecule that specifically binds to a receptor that mediates or is involved in the induction of apoptosis within the cell. In some embodiments, the immune checkpoint regulator may comprise a molecule that specifically binds to a "checkpoint" protein, such as PD1. In some embodiments, the heat shock protein inhibitor may comprise a molecule that specifically binds to a heat shock protein, such as Hsp90. In some embodiments, these molecules can be easily removed during the manufacturing process, for example, by washing the cells in connection with cell manufacturing or prior to final formulation of the cells for administration.

[0157] In some embodiments of the provided methods, a source of potential tumor peptides is used to identify TCRs reactive to neoantigens in a process that includes expanding T cells reactive to tumor neoantigen peptides. The provided methods include an ex vivo co-culture method in which a T cell population expanded from a biological sample (e.g., tumor fragments or peripheral blood or other T cell source) is incubated in the presence of antigen-presenting cells that have been contacted with or presented neoantigen peptides. In certain aspects, the T cells and antigen-presenting cells are autologous to the tumor-bearing subject from whom the peptides were identified. The provided methods further include steps for isolating, enriching, and / or selecting tumor-reactive T cells from the co-culture prior to or in conjunction with additional ex vivo expansion.

[0158] Figure 1A shows a schematic diagram of an exemplary process for manufacturing a T cell therapy composition according to the provided methods. In the exemplary process, a tumor sample is obtained from a patient for identification and generation of peptides for use in co-culture with antigen-presenting cells (APCs) that present the peptides and with autoantigen-associated T cells obtained from the same subject. In some cases, a T cell population obtained from the patient, including, for example, tumor-infiltrating lymphocytes (TILs) or peripheral blood lymphocytes (PBLs), is stimulated under conditions for cell expansion before co-culture with antigen-presenting cells that have been contacted or exposed to peptide neoepitopes for presentation on the major histocompatibility complex. After co-culture under conditions in which the antigen-presenting cells present the peptides in the context of the major histocompatibility complex, tumor-reactive T cells, or T cells surface-positive for one or more T cell activation markers associated with tumor-reactive T cells (also referred to as upregulatory markers or reactive T cell markers, e.g., CD70a), can be selected and cultured under conditions for expansion according to the provided methods, such as incubation with a T cell stimulant (e.g., recombinant IL-2, anti-CD3, and / or anti-CD28). The culturing step can be performed in the presence of one or more recombinant cytokines (e.g., IL-2) to support cell growth and expansion. This process can be performed in the presence of nutrient-containing serum-free medium. One or more, or all, of the steps can be performed in a closed system, e.g., without exposing the cells to the environment. Once a therapeutic dose or threshold number of cells is reached, the cells can be harvested and formulated, optionally concentrated or cryopreserved, and used for administration to a subject, e.g., by infusion. In the provided example, one or more of the steps are performed in the presence of a T cell adjuvant, such as a costimulatory agonist, an apoptosis inhibitor, an immune checkpoint regulator, and / or a heat shock protein inhibitor. Figure 1B shows an exemplary process in which a cryopreservation step can be performed after one or more of the steps.

[0159] The provided methods offer advantages over existing methods for generating expanded TILs because they include a step for enriching tumor-reactive cells, for example, by co-culturing with peptide-presenting APCs followed by selection of reactive T cell clones that upregulate one or more T cell activation markers. This process enriches an initial small population of tumor-reactive T cells expanded from a biological sample (e.g., a tumor) for cells that are or are likely to be tumor-reactive cells prior to a subsequent second expansion step, thereby facilitating the preservation and expansion of cells of interest and limiting the expansion of bystander T cells, which may include cells that are not reactive to tumor antigens and / or exhibit inhibitory activity (Figure 2A). This contrasts with existing methods that involve passive expansion of bulk T cells, in which all tumor-derived T cells are subjected to a first initial expansion, for example, using a high IL-2 concentration, followed by a second rapid expansion of T cells present after the initial expansion. While these alternative processes can significantly expand total viable T cells (TVC), these methods do not actively ensure that tumor-reactive T cells are predominantly expanded (Figure 2B). Furthermore, the provided methods are performed to maximize the number of tumor-reactive cells that can be collected, for example, by co-culturing all cells expanded after a first expansion with peptide-presenting APCs, and then selecting cells positive for one or more activation markers from among the total bulk cells after co-culture before a subsequent second expansion. In aspects of the provided methods, all steps of the method are performed in a closed system.

[0160] The provided methods include one or more features that provide or relate to an improved, more efficient, and / or more robust process for generating tumor-reactive T cell therapeutic compositions ex vivo. In particular, the present disclosure relates to methods that offer advantages over available methods for generating TIL therapeutic cell compositions. Such advantages include, for example, reduced cost, streamlining, improved enrichment of tumor-reactive T cells in the therapeutic composition, and increased efficacy of the therapeutic composition across, for example, a variety of subjects and tumor conditions.

[0161] Among the findings herein is the observation that relatively low concentrations of recombinant IL-2 can be successfully used during one or both expansion stages. Many existing methods use IL-2 at concentrations as high as 6000 IU / mL for T cell expansion of TILs. However, high IL-2 concentrations can increase the cost of the process and can be limiting. In some cases, high IL-2 concentrations can have a negative impact on T cell differentiation by promoting effector T cell differentiation over early memory T cells, which may be more desirable in therapeutic T cell compositions. The provided methods can be performed using concentrations several times lower than 6000 IU / mL, e.g., concentrations less than or about 1000 IU / mL, e.g., from 300 IU / mL or about 300 IU / mL to 1000 IU / mL or about 1000 IU / mL. In certain embodiments, the IL-2 concentration is 300 IU / mL or about 300 IU / mL.

[0162] In embodiments of the provided methods, the T cell population is obtained from a biological sample known to contain T cells. In some embodiments, the T cell population is enriched from a biological sample from a subject, particularly a human subject. The biological sample can be any sample containing a bulk population of T cells. In some embodiments, the biological sample is or comprises peripheral blood mononuclear cells. In some embodiments, the biological sample is a peripheral blood sample or a serum sample. In some embodiments, the biological sample is a lymph node sample. In some embodiments, the biological sample is a tumor sample. In some aspects, the bulk T cells can include tumor-infiltrating T cells (TILs). In some embodiments, the subject is a human subject. In some embodiments, the subject has cancer, a viral infection, a bacterial infection, or an inflammatory condition. In certain embodiments, the subject has cancer.

[0163] In aspects of the provided methods, the starting source of cells in the method can be tumor fragments (e.g., fragments 1-8 mm in diameter) or single-cell suspension preparations obtained from enzymatic digestion of tumor fragments. While certain sources may be superior for some tumor types, it has been found herein that both fragments and single-cell suspensions can support T cell expansion and enrichment of tumor-reactive T cells. In some cases, the tumor cell source can be selected depending on the tumor type or cancer, e.g., to optimize or increase the expansion and enrichment of tumor-reactive T cells from the tumor. In one example, the cancer is melanoma, and the starting population of lymphocytes is, for example, tumor fragments from a resected tumor. In another example, the cancer is colorectal cancer, and the starting population of lymphocytes is a single-cell suspension obtained by enzymatic digestion of tumor fragments, e.g., with collagenase.

[0164] In some embodiments, the method comprises first co-culturing the expanded T cells with peptide-loaded autologous antigen-presenting cells.The findings herein demonstrate that a relatively low concentration of peptide or peptide pool (including multiple peptides, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more, or any value between any of the above), such as when each individual peptide is less than 20 ng / mL, or even as low as 0.1 ng / mL, can result in increased activation of T cells in culture.In some embodiments, this can result in improved enrichment of tumor-reactive T cells in co-culture before selecting cells that are positive for one or more T cell activation markers (i.e., upregulation markers or reactive T cell markers). In some embodiments, the co-culturing step in the provided methods comprises loading the APCs with individual peptides or peptide pools at a ratio of tumor-derived cells containing T cells to autologous APCs (e.g., dendritic cells) from at or about 1:5, to at or about 5:1, e.g., 1:1 or about 1:1, e.g., 1:3, 3:1, or about 3:1. In some embodiments, the APCs are loaded with peptides or peptide pools at a concentration such that the individual peptides or individual peptides of the peptide pool average less than or about 20 ng / mL, e.g., from at or about 0.1 ng / mL to at or about 1 ng / mL, e.g., 0.1 ng / mL or about 0.1 ng / mL.

[0165] In some embodiments, the provided methods include enriching or selecting a T cell population from a biological sample. In some aspects, T cells, or specific subpopulations of T cells, such as cells that are positive for or express high levels of one or more surface markers, e.g., CD3+ T cells, CD4+ T cells, or CD8+ T cells, are isolated by positive or negative selection techniques. In some aspects, the enriched T cells are enriched or selected for CD4+ T cells. In some aspects, the enriched T cells are enriched or selected for CD8+ T cells. In some aspects, the enriched T cells are enriched or selected for CD4+ T cells and CD8+ T cells. For example, CD4+ T cells and CD8+ T cells can be positively selected from bulk T cells that express CD3. Alternatively, CD4+ T cells and CD8+ T cells can be separately selected, either simultaneously or sequentially in either order, by positively selecting for T cell subpopulations that express CD4 and T cell subpopulations that express CD8. Selection of CD4+ and CD8+ T cells ensures enrichment of MHC class II and MHC class I expressing T cells to provide pan-tumor scanning targeted T cell therapy capable of recognizing a diverse repertoire of antigens, including cancer antigens.

[0166] In some embodiments, the methods provided include enriching T cells, e.g., CD3+ T cells, or CD4 and / or CD8 subsets thereof, further based on one or more markers whose expression is upregulated on or specific to reactive or activated T cells (e.g., compared to resting or non-activated T cells) (hereinafter "reactive T cell markers" or T cell activation markers). Reactive T cells express a particular reactive marker when their endogenous TCR recognizes an antigen on a target cell or target tissue, e.g., when the TCR recognizes a neoantigen on a tumor. Exemplary reactive T cell markers include one or more, e.g., two, three, four, or more, of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258, CD256, PD-1, TIM-3, or LAG-3. Enrichment or selection of cells positive for one or more such reactive T cell markers can be performed before or during one or more steps of the expansion method. In certain embodiments, the provided methods involve enrichment or selection of cells positive for one or more upregulated markers on reactive or activated T cells after activation of the T cell population by coculture incubation with peptide-presenting APCs (e.g., dendritic cells, DCs). In some embodiments, selecting cells positive for one or more upregulated markers on reactive or activated T cells from the co-culture can result in a two-fold or greater enrichment of antigen-specific tumor-reactive T cells and / or a substantial reduction in TCR clonality, demonstrating enrichment of TCR clonotypes consistent with enrichment of tumor-reactive T cells. Furthermore, such enriched T cells can exhibit improved ability to produce IFN-γ following antigen-specific stimulation compared to unselected or bulk T cells from the co-culture.

[0167] In some embodiments, the methods generate or expand T cells for use in adoptive cell therapy methods for treating diseases or conditions in which cells or tissues associated with the disease or condition are known or suspected to express an antigenic target recognized by the T cells. In some embodiments, the T cell therapy is autologous to the subject. In some embodiments, the T cell therapy is allogeneic to the subject.

[0168] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0169] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0170] I. Ex vivo expansion methods Provided herein are methods for ex vivo expansion and production of T cell therapy compositions, particularly for use in connection with cancer treatment. In some embodiments, the manufacturing methods involve the propagation and manipulation of patient cells outside the body. In particular embodiments, the methods relate to methods for expanding T cells containing endogenous TCRs specific for tumor-associated antigens (hereinafter "tumor-reactive T cells"). For purposes of this disclosure, reference to tumor-reactive T cells includes T cells that exhibit reactivity to tumor antigens or T cells that are likely or suspected to be tumor-reactive T cells due to upregulation or positive surface expression of T cell activation markers. In some aspects, the frequency of these cells may be low, and ex vivo methods of enrichment and expansion are required to expand these cells to therapeutic doses.

[0171] The provided methods for expanding tumor-reactive T cells include a series of expansion steps to stimulate or induce T cell proliferation within a T cell population. In some cases, the methods include incubating with recombinant IL-2, alone or in combination with one or more other recombinant cytokines (e.g., IL-7, IL-21, and / or IL-15), and optionally one or more other stimulatory T cell agents, to provide the cells with primary and secondary (costimulatory) signals. Additionally, in some cases, one or more T cell modulators can be used, including apoptosis inhibitors and heat shock protein inhibitors and immune checkpoint regulators. A standard method for culturing T cells and providing the cells with primary and secondary (costimulatory) signals includes incubation with T cell stimulatory agents provided by anti-CD3 (e.g., OKT3) and anti-CD28 reagents. In some embodiments, the T cell stimulators include anti-CD3 antibodies (e.g., OKT3) and anti-CD28 antibodies. Typically, such stimulation also includes one or more additional recombinant cytokines (e.g., IL-2, IL-7, IL-21 and / or IL-15) and nutrient-containing medium to enable the cells to survive outside the body.

[0172] In the provided methods, the methods include culturing a T cell population containing tumor-reactive T cells ex vivo, wherein at least a portion of the culturing step includes incubation with an additional T adjuvant comprising a pharmaceutical agonist and, optionally, an inhibitor of an apoptosis- or heat shock protein-mediated pathway. Adding one or more T cell adjuvants to the T cell production process can enhance T cell functionality ex vivo and in vivo. In connection with the provided methods, the methods further include enriching T cells comprising an endogenous TCR specific for a tumor-associated antigen ("tumor-reactive T cells") to maximize the expansion of desired therapeutic cells. In some embodiments, the tumor-associated antigen is or comprises a neoantigen.

[0173] Thus, among the methods provided are those for culturing T cells to produce tumor-reactive T cells that (1) both include using an additional T cell modulatory agent prior to or concurrently with a standard T cell stimulator, such as, for example, anti-CD3 / anti-CD28 and / or recombinant cytokines (e.g., IL-2, IL-7, IL-21, and / or IL-15), and (2) further include enrichment or selection for tumor-reactive T cells or T cells that are surface positive for one or more T cell activation markers associated with tumor-reactive T cells. It is believed that the provided methods can increase expansion to therapeutic doses and / or enhance the functionality of T cell therapy for therapeutic efficacy to a much greater extent than existing methods.

[0174] The provided methods include collecting a biological sample from a subject that is known to contain tumor-reactive T cells or is likely to contain tumor-reactive T cells. In some embodiments, the biological sample can be collected directly from a tumor-bearing subject; in some cases, such isolated or obtained T cells may be co-cultured with or exposed to a tumor in vivo. In embodiments of the provided methods, the T cell-containing population (hereinafter also referred to as a first T cell population) is a T cell population obtained, selected, or isolated from a biological sample from a subject, such as a human subject. In some embodiments, the T cell-containing population can be from any source sample known or suspected to contain T cells that are tumor-reactive T cells or that may or may potentially contain tumor-reactive T cells. The sample can include a tumor sample containing tumor-infiltrating lymphocytes (TILs), a blood sample containing peripheral blood mononuclear cells (PBMCs) (e.g., an apheresis sample or leukapheresis sample), or a lymph node sample. In some embodiments, the sample is a tumor sample or tumor fragment containing tumor-infiltrating lymphocytes or TILs. A population containing T cells can be obtained directly from a subject (e.g., a healthy subject or a subject with cancer), for example, by selecting T cells or a subset thereof from a biological sample from the subject. In certain embodiments, the biological sample is derived from a subject having a tumor and potentially containing tumor-reactive T cells, or potentially containing tumor-reactive T cells that can be enriched by the provided methods, or may contain tumor-reactive T cells that can be enriched by the provided methods.

[0175] The provided methods for expanding tumor-reactive T cells include a first expansion step that involves culturing a selected or isolated population containing T cells (i.e., a first T cell population) with recombinant cytokines, typically including recombinant IL-2 (e.g., IL-2, IL-7, IL-21, and / or IL-15). In some cases, the T cell stimulator further provides the cells with primary and secondary (co-stimulatory) signals, for example, provided by an anti-CD3 (e.g., OKT3) reagent and an anti-CD28 reagent, e.g., an anti-CD3 antibody (e.g., OKT3) and an anti-CD28 antibody. In some cases, the first expansion step is also performed in the presence of one or more T cell modulators, e.g., a TNFSFR agonist and / or an immune checkpoint regulator and / or an apoptosis inhibitor and / or a heat shock protein inhibitor, as described. The initial or first expansion step results in a second T cell population enriched for T cells as a result of the expansion or proliferation of T cells present in the first population.

[0176] In other embodiments of the provided methods, a biological sample can be collected and used for ex vivo co-culture, in which T cells obtained directly from a subject (e.g., a healthy subject or a cancer subject) are incubated with artificial presentation cells (APCs) under conditions in which the APCs are induced to present one or more peptides derived from a tumor-associated antigen from the subject. The collected sample contains lymphocytes with endogenous TCRs reactive to mutations present on the tumor. These cells can be identified by various methods, including, but not limited to, co-culture in the presence of antigen-presenting cells or co-culture with the tumor. In the provided methods, tumor-reactive T cells can be further identified or enriched from the stimulated T cells expanded in the first step by one or more additional steps, including ex vivo co-culture of the stimulated or expanded T cells (i.e., a second T cell population) with antigen-presenting cells and one or more peptides containing neoepitopes of the tumor antigen (APC / peptide neoepitope). The provided methods include ex vivo co-culture in which a second T cell population is incubated with artificial presentation cells (APCs) that have been exposed to or contacted with one or more peptides, e.g., synthetic peptides, under conditions in which the APCs are induced to present one or more peptides derived from tumor-associated antigens. In some embodiments, the population T cells are autologous T cells from a tumor-bearing subject, and the source of the synthetic peptides is a tumor antigen peptide derived from a tumor antigen of the same subject. In some embodiments, the cells obtained from the ex vivo co-culture include a T cell population (optionally a third T cell population) that is a source of cells enriched for tumor-reactive T cells. Optionally, co-culture of T cells with APCs and peptides can also be performed in the presence of one or more T cell modulators (e.g., TNFSFR agonists and / or apoptosis inhibitors).

[0177] In some cases, tumor-reactive T cells can be further enriched by separating or selecting cells that express one or more activation markers associated with tumor-reactive T cells. Among the provided aspects, biological samples containing tumor-reactive T cells, including samples obtained directly from subjects or ex vivo-generated co-culture samples, can be further enriched for tumor-reactive T cells or T cells that express one or more activation markers associated with tumor-reactive T cells. T cell activation markers include cell surface markers whose expression is upregulated or specific to T cells that have been exposed to an antigen and activated. Examples of such markers are described below. In the provided aspects, a T cell population containing tumor-reactive T cells is isolated, selected, or enriched from a biological sample before or in conjunction with expanding such cells according to the provided methods. In some embodiments, tumor-reactive T cells or T cells that express specific activation markers associated with tumor-reactive T cells are endogenously present in cancer subjects known to have tumors. In other embodiments, tumor-reactive T cells, or T cells expressing specific activation markers associated with tumor-reactive T cells, are obtained after ex vivo co-culture of autologous T cells from a tumor-bearing subject with a source of tumor antigen peptides under conditions that allow tumor-reactive T cells to be generated and identified.

[0178] In certain embodiments, the enriched or isolated T cells from the coculture are subjected to a second expansion, such as after separation or selection of tumor-reactive T cells or T cells surface-positive for one or more T cell activation markers associated with tumor-reactive T cells. The second expansion includes incubation with a T cell stimulator, such as an anti-CD3 antibody (e.g., OKT3), an anti-CD28 antibody, and a recombinant cytokine (e.g., IL-2, IL-7, IL-21, and / or IL-15), and optionally one or more T cell modulators (e.g., a costimulatory agonist and / or an immune checkpoint regulator and / or an apoptosis inhibitor and / or a heat shock protein inhibitor, as described), to further stimulate the T cells. The T cells, e.g., tumor-reactive T cells or T cells surface-positive for one or more T cell activation markers associated with tumor-reactive T cells, can be expanded for a number of days as desired and / or until a therapeutic dose or harvest dose is met. A composition of the expanded T cells can then be harvested and formulated for administration to a subject for treatment of the subject's cancer.

[0179] Provided herein are methods for producing tumor-reactive T cells, wherein the cells are cultured ex vivo by a process comprising incubating a T cell population containing, or likely to contain, tumor-reactive T cells comprising an endogenous TCR reactive to a tumor-associated antigen in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, at least a portion of which is carried out before, simultaneously with, or after incubating the T cell population with a T cell stimulator, e.g., an anti-CD3 antibody (e.g., OKT3), an anti-CD28 antibody, and / or a recombinant cytokine (e.g., IL-2, IL-7, IL-21, and / or IL-15), under conditions to stimulate expansion of the cells of the T cell population. The described T cell adjuvants, e.g., costimulatory agonists and / or immune checkpoint regulators and / or apoptosis inhibitors and / or heat shock protein inhibitors, can be included during either or both of the first expansion or the second expansion according to the provided methods. In some embodiments, the culturing method includes the steps of: (a) incubating an ex vivo population of T cells, including tumor-reactive T cells comprising endogenous TCRs reactive to a tumor-associated antigen, with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; and (b) incubating the T cell population with a T cell stimulator under conditions to stimulate proliferation of cells of the cell population, wherein, for example, in either or both of the first or second expansion, at least a portion of the incubating steps to stimulate or expand the T cells occurs before, simultaneously with, or after the incubating step with at least one T cell adjuvant. In some embodiments, the T cell population incubated in the presence of at least one T cell adjuvant can be a first T cell population after obtaining, selecting, or isolating the T cell population from a biological sample from a subject. In embodiments, the T cell population is enriched for CD4+ T cells and CD8+ T cells.In some embodiments, tumor-reactive T cells can be enriched from a biological sample containing T cells prior to incubating with at least one T cell adjuvant and / or prior to incubating with at least one T cell stimulator. In some embodiments, the T cell population incubated in the presence of at least one T cell adjuvant can be a T cell population (e.g., optionally a fourth population of T cells) that has been enriched or selected for tumor-reactive T cells or T cells expressing activation markers after co-culture with APC / peptide neoepitopes. In some embodiments, the method includes culturing the cells under expansion conditions until a threshold amount of cells is obtained and / or up to 20 days after initiation of incubation with at least one T cell adjuvant, wherein at least a portion of the culturing occurs during incubation with one or more T cell adjuvants and / or T cell stimulators.

[0180] Provided herein are methods for producing tumor-reactive T cells, in which cells are cultured ex vivo by a process including the steps of: enriching a sample containing T cells for tumor-reactive T cells that contain an endogenous TCR that is reactive to a tumor-associated antigen, thereby generating a T cell population enriched in tumor-reactive T cells; and incubating the T cell population enriched in tumor-reactive T cells in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, wherein at least a portion of the incubation occurs before, concurrently with, or after incubating the T cell population with a T cell stimulator, e.g., an anti-CD3 antibody (e.g., OKT3), an anti-CD28 antibody, and / or a recombinant cytokine (e.g., IL-2, IL-7, IL-21, and / or IL-15), under conditions to stimulate expansion of cells of the T cell population. In some embodiments, tumor-reactive T cells are selected or isolated after co-culturing a T cell population (and optionally a second T cell population) containing T cells stimulated in the presence of antigen-presenting cells (APCs) that have been contacted or exposed to one or more of the plurality of peptides under conditions in which the APCs present one or more MHC-associated non-native peptides. In some embodiments, the culture method includes the steps of: (a) enriching tumor-reactive T cells comprising an endogenous TCR reactive to a tumor-associated antigen from a biological sample containing T cells, e.g., from a co-culture of T cells with an APC / neoepitope peptide, thereby generating a T cell population enriched for tumor-reactive T cells; (b) incubating the tumor-reactive T cell-enriched T cell population with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; and (c) incubating the tumor-reactive T cell-enriched T cell population with a T cell stimulator under conditions to stimulate expansion of cells of the cell population, wherein at least a portion of the incubating with the T cell stimulator occurs before, simultaneously with, or after the incubating with the at least one T cell adjuvant. In embodiments, the T cell population is enriched for CD4+ T cells and CD8+ T cells.In some embodiments, the method comprises culturing the cells under conditions for expansion until a threshold amount of cells is obtained and / or up to 20 days after initiation of incubation with at least one T cell adjuvant, wherein at least a portion of the culturing occurs during incubation with one or more T cell adjuvants and / or incubation with a T cell stimulator.

[0181] Provided herein are methods for producing tumor-reactive T cells, in which the cells are cultured ex vivo by a process comprising: incubating a T cell population with a T cell stimulatory agent, e.g., a recombinant cytokine from IL-2, IL-7, IL-21, and / or IL-15, such as generally including at least recombinant IL-2, and at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, wherein the incubation is carried out under conditions that stimulate or expand T cells in the population to generate a second T cell population; enriching the T cell population for tumor-reactive T cells that contain an endogenous TCR that is reactive to a tumor-associated antigen, thereby generating a T cell population enriched for tumor-reactive T cells; and incubating the T cell population enriched for tumor-reactive T cells with a T cell stimulatory agent, e.g., an anti-CD3 antibody (e.g., OKT3), an anti-CD28 antibody, and a recombinant cytokine (e.g., IL-2, IL-7, IL-21, and / or IL-15), under conditions to further stimulate expansion of cells of the tumor-reactive T cell-enriched cell population. In some embodiments, tumor-reactive T cells are selected or isolated after co-culturing a T cell population (and optionally a second T cell population) containing T cells stimulated in the presence of antigen-presenting cells (APCs) that have been contacted or exposed to one or more of the plurality of peptides under conditions in which the APCs present one or more MHC-associated non-native peptides. In embodiments, the T cell population is enriched for CD4+ T cells and CD8+ T cells. In some embodiments, the method comprises culturing the cells under conditions for expansion until a threshold amount of cells is obtained and / or for up to 20 days after initiation of incubation with at least one T cell adjuvant, wherein at least a portion of the culturing occurs during incubation with one or more T cell adjuvants and / or incubation with a T cell stimulator.

[0182] In certain embodiments, the provided methods include, but are not limited to, (1) identifying, obtaining, or generating a plurality of peptides comprising neoepitopes specific to a tumor of a subject; (2) obtaining a T cell population (first T cell population) from a donor subject, e.g., obtained from a resected tumor or obtained by directly selecting T cells from a biological sample, e.g., tumor, blood, bone marrow, lymph node, thymus, or other tissue or fluid; (3) administering a T cell stimulator, such as one or more recombinant cytokines from IL-2, IL-7, IL-21, and / or IL-15 (e.g., including at least recombinant IL-2), and optionally one or more T cell stimulators, such as a TNFRSF agonist and / or an apoptosis inhibitor. (3) co-culturing the second population containing the stimulated T cells in the presence of antigen-presenting cells (APCs) that have been contacted or exposed to one or more of the plurality of peptides, under conditions in which the APCs present one or more MHC-associated non-native peptides, to generate a third T cell population; and (5) enriching the third T cell population for T cells containing endogenous TCRs reactive to peptides present on the antigen-presenting cells (APCs) to generate a fourth T cell population. In some aspects, T cells containing endogenous TCRs are enriched by separating antigen-presenting cells from the T cell population. Alternatively or additionally, such cells are enriched by selecting T cells that are surface-positive for one or more activation markers associated with tumor-reactive T cells. In certain embodiments, the enriched or isolated T cells from the co-culture are subjected to a second expansion, such as after separation or selection of tumor-reactive T cells or T cells that are surface positive for one or more T cell activation markers associated with tumor-reactive T cells.The second expansion involves incubation to further stimulate the T cells with T cell stimulators, such as anti-CD3 antibodies (e.g., OKT3), anti-CD28 antibodies, and recombinant cytokines (e.g., IL-2, IL-7, IL-21, and / or IL-15), and optionally one or more T cell modulators (e.g., TNFSFR agonists and / or immune checkpoint modulators and / or apoptosis inhibitors and / or heat shock protein inhibitors).

[0183] In embodiments of the provided methods, one or more of the steps can be performed in serum-free medium. In one embodiment, the serum-free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcell technologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline (Sigma), Xvivo15 (Lonza), PrimeXV (Irvine Scientific), or Stem XVivo (RandD systems). The serum-free medium can be supplemented with a serum substitute, such as ICSR (immune cell serum replacement) from LifeTech. The level of serum substitute (e.g., ICSR) can be, for example, up to 5%, e.g., about 1%, about 2%, about 3%, about 4%, or about 5%. In some embodiments, the serum-free medium contains 0.5 mM to 5 mM of a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine (Glutamax™). In some embodiments, the concentration of the dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine, is 0.5 mM to 5 mM, 0.5 mM to 4 mM, 0.5 mM to 3 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, 1 mM to 4 mM, 1 mM to 3 mM, 1 mM to 2 mM, 2 mM to 5 mM, 2 mM to 4 mM, 2 mM to 3 mM, 3 mM to 5 mM, 3 mM to 4 mM, or In some embodiments, the concentration of a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine, is 2 mM or about 2 mM.

[0184] In any of the embodiments of the provided methods, incubation with each of the at least one T cell adjuvant, e.g., one or more costimulatory agonists, immune checkpoint modulators, heat shock protein inhibitors, or apoptosis inhibitors, is independently continued throughout the entire course of the culturing step, or a portion thereof. In some embodiments, incubation with each of the at least one T cell adjuvant is for 14 days or less, 12 days or less, 10 days or less, 7 days or less, 5 days or less, 3 days or less, or 2 days or less. In some embodiments, incubation with each of the at least one T cell adjuvant is independently for 12 hours to 96 hours, e.g., 24 hours to 48 hours, typically 48 hours or about 48 hours.

[0185] In embodiments, incubation with each of the T cell stimulators, such as recombinant cytokines (e.g., IL-2) and / or anti-CD3 / anti-CD28 antibodies, can be continued for a period sufficient to activate or stimulate the cells. In some embodiments, incubation with the T cell stimulator is carried out for 1 day or about 1 day, e.g., generally 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days, or for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days, or any range of time between any of the foregoing. In some embodiments, incubation is carried out for 7-10 days. In some embodiments, incubation is for 7 days or about 7 days. In some embodiments, incubation is for 8 days or about 8 days. In some embodiments, incubation is for 9 days or about 9 days. In some embodiments, incubation is for 10 days or about 10 days. In some embodiments, incubation with each of the T cell stimulants, such as recombinant cytokines (e.g., IL-2) and / or anti-CD3 / anti-CD28 antibodies, is for 12 to 96 hours, e.g., 24 to 48 hours, typically 48 hours or about 48 hours.

[0186] In some embodiments, the cells are washed one or more times during culture to remove agents present in the culture and / or to supplement the culture medium with one or more additional agents, hi some embodiments, the cells are washed during culture to reduce or remove at least one T cell adjuvant and / or at least one T cell stimulator prior to completion of culture.

[0187] In some embodiments, the T cell culture methods provided herein, including incubation with a T cell adjuvant and / or a T cell stimulant, include a temperature suitable for the proliferation of human T lymphocytes, e.g., at least about 25° C., generally at least about 30° C., generally at or about 37° C. In some embodiments, the culture methods, including incubation with a T cell adjuvant and / or a T cell stimulant, are performed in serum-free medium.

[0188] In certain embodiments, the provided methods include enriching from a biological sample (either sourced directly from the sample in vivo or derived from ex vivo co-culture with antigen-presenting cell (APC) T cells bearing an endogenous TCR that recognizes a tumor-associated antigen, e.g., a neoantigen, such as by selecting for T cells that are surface positive for one or more T cell activation markers (e.g., CD107, CD107a, CD039, CD137, CD59, CD90, CD38, or CD103).

[0189] In some embodiments, any one or more of the method steps can be performed in a closed system or under GMP conditions. In certain embodiments, all process operations are performed in a GMP suite. In some embodiments, a closed system is used to perform one or more of the other processing steps of the method for manufacturing, producing, or producing a cell therapy. In some embodiments, one or more or all of the processing steps, such as isolation, selection and / or enrichment, processing, culture steps including incubation associated with cell expansion, and formulation steps, are performed in an integrated or self-contained system and / or using a system, device, or instrument in an automated or programmable manner. In some embodiments, the system or instrument includes a computer and / or computer program that communicates with the system or instrument, allowing a user to program and control various aspects of the processing, isolation, manipulation, and formulation steps, evaluate the results of various aspects of the processing, isolation, manipulation, and formulation steps, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0190] In some embodiments, the culturing method for expanding cells according to any of the provided methods is carried out until a threshold amount of cells, e.g., tumor-reactive cells or cells positive for one or more T cell activation markers, is obtained, and / or for up to 20 days after initiation of incubation with at least one T cell adjuvant. In some embodiments, the culturing step is carried out for 7-20 days, 7-14 days, 7-10 days, 10-20 days, 10-14 days, or 14-20 days.

[0191] In some embodiments, the culturing step is performed until a threshold amount of cells is obtained, the threshold amount being 0.5×10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 30 x 10 9pieces or approximately 30 x 10 9 0.5 x 10 total cells or total viable cells 8 From 12 x 10 9 pieces or approximately 12 x 10 9 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 0.5 x 10 total cells or total viable cells 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 1 x 10 whole cells or total viable cells 8 From 50 x 10 9 or approximately 50 x 10 9 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 1 x 10 whole cells or total viable cells 8 From 12 x 10 9 pieces or approximately 12 x 10 9 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 1 x 10 whole cells or total viable cells8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 1 x 10 whole cells or total viable cells 8 or approximately 1 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 3.5 x 10 total cells or total viable cells 8 pieces or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 98 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 8 x 10 total cells or total viable cells 8 pieces or approximately 8 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 15 x 10 total cells or total viable cells 8 pieces or approximately 15 x 10 8 From 60 x 10 8 pieces or approximately 60 x 10 8 60 x 10 whole cells or total viable cells 8 pieces or approximately 60 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 60 x 10 whole cells or total viable cells 8 pieces or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 109 60 x 10 whole cells or total viable cells 8 pieces or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 12 x 10 total cells or total viable cells 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 12 x 10 total cells or total viable cells 9 pieces or approximately 12 x 10 9 From 30 x 10 9 pieces or approximately 30 x 10 9 total or viable cells, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 pieces or approximately 60 x 10 9 total cells or total live cells (inclusive).

[0192] In some of any of the provided embodiments, the method results in a fold expansion of T cells or a fold expansion of tumor-reactive T cells that is at least or at least about 2-fold, at least or at least about 5-fold, at least or at least about 10-fold, at least or at least about 25-fold, at least or at least about 25-fold, at least or at least about 50-fold, at least or at least about 100-fold, at least or at least about 250-fold, at least or at least about 500-fold, at least or at least about 1000-fold, or more.

[0193] The following subsections further describe non-limiting descriptions of aspects of the provided methods.

[0194] A. Neoepitope Identification and Peptide Generation The provided methods include generating or identifying in silico a plurality of peptides (also referred to as "P" or "n-mers") comprising at least one cancer-specific cancer neoepitope, and further filtering the peptides in silico to obtain a subset of neoepitope sequences. In some embodiments, at least one synthetic peptide is prepared using sequence information from the subset of neoepitope sequences, and the synthetic peptide is then used in a method for enriching tumor-reactive T cells according to the provided methods.

[0195] In some embodiments, the method for ex vivo generation of tumor-reactive T cells comprises identifying or isolating tumor-associated antigens or their peptide sequences from cancer cells from a subject.Cancer cells can be obtained from any body sample from a patient that contains or is expected to contain tumor cells or cancer cells.The body sample can be any tissue sample, such as blood, tissue sample obtained from primary tumor or tumor metastasis, lymph node sample, or any other sample containing tumor cells or cancer cells.In some aspects, nucleic acids from such cancer cells are obtained and sequenced.In embodiments, the protein-coding regions of genes in the genome are sequenced by whole exome sequencing or the like.To identify tumor-specific sequences, the sequencing data can be compared with reference sequencing data, for example, data obtained by sequencing normal cells or non-cancerous cells from the same subject.In some embodiments, next-generation sequencing (NGS) methods are used.

[0196] In some embodiments, the tumor is a hematological tumor.Non-limiting examples of hematological tumors include leukemia, for example, acute leukemia (such as l lq23 positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0197] In some embodiments, the tumor is a solid tumor. Non-limiting examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma, and lobular carcinoma of the breast), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, and thyroid carcinoma. carcinoma), papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some examples, the tumor is melanoma, lung cancer, lymphoma, breast cancer, or colon cancer.

[0198] In some embodiments, the cancer is a cancer of the gastrointestinal (GI tract), e.g., cancer, or GI cancer involving the upper or lower GI tract, or accessory organs of digestion, such as the esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum, or anus. In some embodiments, the cancer is esophageal cancer, stomach (gastric) cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, cancer of mucosa-associated lymphoid tissue (MALT lymphoma), cancer of the bile duct, colorectal cancer (including colon cancer, rectal cancer, or both), anal cancer, or GI carcinoid tumor. In particular embodiments, the cancer is colorectal cancer.

[0199] In some embodiments, the tumor originates from a breast cancer, such as ductal carcinoma or lobular carcinoma. In some embodiments, the tumor originates from prostate cancer. In some embodiments, the tumor originates from a skin cancer, such as basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, or melanoma. In some embodiments, the tumor originates from a lung cancer, such as adenocarcinoma, bronchioloalveolar carcinoma, large cell carcinoma, or small cell carcinoma. In some embodiments, the tumor originates from a brain cancer, such as glioblastoma or meningioma. In some embodiments, the tumor originates from a gastrointestinal cancer, such as any of the above. In some embodiments, the tumor originates from colon cancer. In some embodiments, the tumor originates from a liver cancer, such as hepatocellular carcinoma. In some embodiments, the tumor originates from pancreatic cancer. In some embodiments, the tumor originates from a kidney cancer, such as renal cell carcinoma. In some embodiments, the tumor originates from testicular cancer.

[0200] In some embodiments, the cancer is not melanoma. Melanoma is a cancer that generally has a high mutation rate. A high tumor mutation burden has been considered a particularly desirable prognostic marker for the success of immunotherapy treatment targeting tumor neoantigens (Simpson et al., Journal of Clinical Oncology 2017, 35:15 Suppl., 9567-9567; McGranahan et al. Science 2016, 351:1463-1469). In some embodiments, the provided method is performed to actively (rather than passively) enrich tumor-reactive T cells, and therefore can be used for cancers with a relatively low tumor mutation burden.

[0201] In some embodiments, the subject has a tumor mutation burden (TMB) of less than 8 mutations. TMB includes the number of nonsynonymous mutations per tumor. In some embodiments, TMB can be calculated by counting the number of synonymous and nonsynonymous mutations over a 0.8-1.2 megabase (Mb) region and reporting the results as mutations / Mb. In some embodiments, TMB can be determined by next-generation sequencing (NGS) of tumor tissue samples. In some cases, whole-exome sequencing can be used, or computational germline status filtering can be used (Chalmers et al. Genome Med 2017 9:34). In some embodiments, the subject has a TMB of less than or about 60 mutations / Mb, e.g., less than or about 55 mutations / Mb, less than or about 55 mutations / Mb, less than or about 50 mutations / Mb, less than or about 45 mutations / Mb, less than or about 40 mutations / Mb, less than or about 30 mutations / Mb, less than or about 25 mutations / Mb, or less than or about 20 mutations / Mb, or any value between any of the foregoing. In some embodiments, the subject has a TMB of less than or about 41 mutations / Mb, less than or about 41 mutations / Mb, less than or about 40 mutations / Mb, less than or about 39 mutations / Mb, less than or about 38 mutations / Mb, less than or about 37 mutations / Mb, or less.

[0202] In some embodiments, the peptide (P) is a tumor-associated antigen derived from a variant of intraepithelial carcinoma or a pre-malignant condition such as vulvar intraepithelial neoplasia, cervical intraepithelial neoplasia, or vaginal intraepithelial neoplasia.

[0203] In some aspects, the nucleic acid from such tumor or cancer cell is obtained and sequenced.In some embodiments, the protein coding region of gene in genome is obtained by, for example, omics analysis, for example, by analyzing whole genome sequencing data, exome sequencing data and / or transcriptome data.To identify tumor-specific sequence, sequencing data can be compared with reference sequencing data, for example, the data obtained from normal cell or non-cancerous cell from the same subject.In some embodiments, next-generation sequencing (NGS) method is used.

[0204] In some embodiments, the method comprises using matched normal omics data of tumor. In such methods, the in silico analysis comprises omics analysis to identify mutations in tumor compared with normal tissue of the same patient, for example, non-disease tissue of the same patient. The matched normal omics data is whole genome sequencing data, exome sequencing data and / or transcriptome data, and the matched normal omics data is generally considered to be matched with normal before the patient's treatment. In certain embodiments, whole exome sequencing is performed on healthy tissue and diseased tissue to identify somatic mutations associated with tumor.

[0205] In some embodiments, the omics data is obtained from one or more patient biopsy samples according to standard tissue processing and sequencing protocols. In certain embodiments, the data is patient-matched tumor data (e.g., tumor vs. normal from the same patient). In some cases, unmatched or matched data to other references (e.g., previous normals from the same patient, or previous tumors from the same patient, or homostatistics) are also considered suitable for use herein. The omics data can be new omics data or omics data obtained from a previous procedure (or a different patient). For example, neoepitopes can be identified in a first stage from a patient tumor by whole genome and / or exome analysis of a tumor biopsy (or lymphoid biopsy, or biopsy from a metastatic site) and matched normal tissue (i.e., non-diseased tissue obtained from the same patient, such as peripheral blood). In some embodiments, genomic analysis can be processed through position-guided synchronous comparison of the omics information thus obtained.

[0206] Genome analysis can be performed by any number of analytical methods. In certain embodiments, the method includes WGS (whole genome sequencing) and exome sequencing of both tumor and matched normal samples using next-generation sequencing such as massively parallel sequencing, ion torrent sequencing, and pyrosequencing. Computational analysis of sequence data can be performed in many ways. In some embodiments, the data format is SAM, BAM, GAR, or VCF format. As an example, analysis can be performed in silico by position-guided synchronous alignment of tumor and normal samples, for example, as disclosed in US Patent No. 2012 / 0059670 and US Patent No. 2012 / 0066001, using BAM files and a BAM server. Alternative file formats for sequence analysis (e.g., SAM, GAR, FASTA, etc.) are also contemplated.

[0207] In some optional embodiments, peptides (P) containing neoantigens resulting from missense mutations include amino acid changes encoded by one or more nucleotide polymorphisms. Peptides (P) containing neoantigens resulting from frameshift mutations, splice site variants, insertions, inversions, and deletions should include novel peptide sequences and junctions of novel peptide sequences. Peptides (P) containing neoantigens with novel post-translational modifications should include amino acids with post-translational modifications such as phosphates or glycans.

[0208] Once these mutations are identified, neoepitopes are then identified. Neoepitopes are mutant peptides recognized by the patient's T cells. These neoepitopes must be presented by tumors or antigen-presenting cells via the MHC complex and then recognized by TCRs on T cells. In some embodiments, the provided methods include calculating one or more neoepitopes to define tumor- and patient-specific neoepitopes. Thus, it should be appreciated that patient- and cancer-specific neoepitopes can be identified exclusively in silico from omics information that ultimately predicts potential epitopes specific to a patient and tumor type. In certain aspects, such identified cancer neoepitopes are unique to the patient and the patient's particular cancer (e.g., having a frequency of less than 0.1% of all neoepitopes, and more typically less than 0.01% within a population of cancer patients diagnosed with the same cancer), but such identified cancer neoepitopes are likely to be presented in tumors.

[0209] In some optional embodiments, the length of the peptide (P) depends on the specific application and is typically about 5 to about 50 amino acids. In preferred embodiments, the peptide (P) is about 7 to 35 amino acids, e.g., 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, or 35 amino acids. In some aspects, the method can be performed using individual peptides containing amino acid sequence variations (e.g., mutations). In some aspects, the method can be performed using a peptide pool, where the peptides in the pool contain amino acid sequence variations (e.g., mutations). The peptide pool can include tens to hundreds of individual peptides. In some cases, a peptide pool may contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more individual peptides, or any value between any of the foregoing. A peptide pool may represent one neo-antigen, or it may represent several neo-antigens. In some cases, a peptide pool may contain multiple overlapping peptides of the same neo-antigen. Thus, in the case of a tumor-associated antigen, the antigen may be divided into 7-35, e.g., 25-amino acid peptides (P), where each peptide (P) contains a unique amino acid composition, or the peptides (P) may be an overlapping peptide pool in which the antigen is divided into a set number of 7-35, e.g., 25-amino acid peptides (P) with overlapping sequences. For example, an overlapping peptide pool comprising a 100 amino acid antigen can be divided into eight 25 amino acid peptides (P), each offset by 12 amino acids (i.e., each subsequent 25 amino acid peptide comprising the 100 amino acid peptide sequence begins 13 amino acid positions from the previous peptide). Those skilled in the art will appreciate that there are many permutations for generating peptide pools from an antigen.

[0210] Neoepitope sequences contemplated herein can be defined as relatively short (e.g., 5-30 mers, more typically 7-11 mers or 12-25 mers) sequence stretches that contain amino acid sequence changes (e.g., mutations). Most typically, the changes are located at or near the center (e.g., less than 4, 5, or 6 amino acids from the central position). In certain aspects, neoepitope sequences contemplated herein particularly include those in which a single amino acid is exchanged relative to the matched normal sequence, with the position of the changed amino acid being located at or near the center of the neoepitope sequence (e.g., in a 9-mer, the changed amino acid is at position 2, 3, 4, or 5, more typically at position 3, 4, or 5, and most typically at position 4 or 5). It should be understood that a single amino acid change can be represented in multiple neoepitope sequences containing the changed amino acid, depending on the position of the changed amino acid.

[0211] In certain embodiments, neoepitopes are calculated to have a length of 2 to 50 amino acids, more typically 5 to 30 amino acids, and most typically 9 to 15 amino acids. For example, when an epitope is presented by the MHC-I complex, the typical epitope length is about 8 to 11 amino acids, whereas a typical epitope for presentation via the MHC-II complex has a length of about 13 to 17 amino acids. As will be readily appreciated, the actual peptide sequence and topology of the neoepitope may vary considerably, as the position of the altered amino acid may be non-centric. Furthermore, when the neoepitope is presented to immunocompetent (or other) cells as a synthetic peptide, it should be understood that the synthetic peptide may be significantly longer than the portion of the peptide ultimately bound by the MHC-I or MHC-II system to allow for intracellular proteolytic processing. Thus, for example, contemplated synthetic peptides may have 8 to 15 amino acids upstream and downstream of the altered amino acid.

[0212] Various algorithms have been developed and can be used to map T cell epitopes (both MHC class I and MHC class II restricted) within protein molecules of various origins. In some embodiments, many programs utilize the availability of large-scale peptide-MHC binding affinity matrices from experimental measurements to train machine learning (ML)-based classifiers to distinguish MHC-binders from non-binders (see, e.g., Zhao et al. (2018) PLoS Comput Biol 14(11):e1006457). Exemplary prediction methods for MHC class I (e.g., 9-mer) include smm, smmpmbec, ann(NetMHC3.4), NetMHC4, PickPocket, consensus, NetMHCpan2.8, NetMHCpan3, NetMHCpan4, NetMHCcons, mhcflurry, mhcflurry_pan, or MixMHCpred. Exemplary prediction methods for MHC class II (e.g., 15-mer) include NetMHCIIpan, NetMHCII2.3, nn_align, smm_align, consensus, comblib, tepitope, or mhcflurry. Any of these methods can be used.

[0213] In embodiments where the synthetic peptide is used for direct MHC-I binding, the total length is 8-10 amino acids. In embodiments where the synthetic peptide is used for direct MHC-II binding, the total length is 12-25 amino acids, e.g., 14-20 amino acids. Optionally, where the synthetic peptide is processed intracellularly (typically via proteasomal processing) prior to MHC presentation, the total length is typically 10-40 amino acids, with the altered amino acid being at or near the center of the synthetic peptide. In some embodiments, the peptide for MHC-I binding is a 9-mer. In some embodiments, the peptide for MHC-II binding is a 23-mer. In some embodiments, the peptide for MHC-II binding is a 25-mer.

[0214] For example, peptide (P) may contain 0 to 25 amino acids on either side of a novel junction resulting from an amino acid change or mutation. In one embodiment, peptide (P) is a neo-antigen sequence, e.g., a 25-amino acid peptide, containing 12 amino acids on either side of an amino acid change resulting from a single nucleotide polymorphism, with the 13th amino acid being the amino acid residue resulting from the single nucleotide polymorphism. In some embodiments, peptide (P) is a neo-antigen sequence, e.g., a 25-amino acid peptide, containing 12 amino acids on either side of an amino acid having a novel post-translational modification, with the 13th amino acid being the amino acid residue resulting from the novel post-translational modification site. In other embodiments, peptide (P) is a neo-antigen sequence containing 0 to 12 amino acids on either side of a novel junction created by an insertion, deletion, or inversion. In some cases, peptide (P) containing a neo-antigen resulting from a novel sequence may encompass the entire novel sequence, including 0 to 25 amino acids on either side of a novel junction that may also result.

[0215] In some embodiments, the sequence differences so identified may be subjected to additional downstream analysis to identify those resulting in new peptide sequences based on cancer- and patient-specific mutations. Thus, neoepitopes can be identified by considering the type of mutation (e.g., deletion, insertion, transversion, transition, translocation) and effect (e.g., nonsense, missense, frameshift, etc.), and thus can act as a content filter to filter out silent and other unrelated (e.g., non-expressing) mutations.

[0216] In some embodiments, the identified neoepitopes can be further filtered in silico against the identified patient HLA type. Such HLA matching is believed to ensure strong binding of the neoepitopes to the MHC-I complex of nucleated cells and the MHC-II complex of specific antigen-presenting cells. Targeting both antigen-presenting systems is particularly believed to result in therapeutically effective and durable immune responses involving both the cellular and humoral branches of the immune system. It should also be understood that the HLA-matched neoepitopes identified in this manner can be biochemically validated in vitro.

[0217] Various methods can be used to perform HLA determination for both MHC-I and MHC-II. In some embodiments, HLA types can be predicted in silico from omics data using reference sequences that include most or all of the known and / or common HLA types. For example, the patient's HLA type is confirmed (using wet chemistry or in silico determination), and the structural solution for the HLA type is calculated or obtained from a database, and then the database is used as an in silico docking model to determine the binding affinity of neoepitopes to the HLA structural solution. Suitable systems for determining binding affinity include the NetMHC platform (see, for example, Nucleic Acids Res. 2008 Jul 1;36 (Web Server Publication):W509-W512), HLAMatchmaker (http: / / www.epitopes.net / downloads.html) and IEDB Analysis Resource (http: / / tools.immuneepitope.org / mhcii / ). Neoepitopes with high affinity for the previously determined HLA types (e.g., less than 100 nM, less than 75 nM, less than 50 nM for MHC-I; less than 500 nM, less than 300 nM, less than 100 nM for MHC-II) are then selected. When calculating the highest affinity, modifications to the neoepitope can be performed by adding N- and / or C-terminal modifications to the epitope to further increase binding of the synthetic neoepitope to the patient's HLA type. Thus, the neoepitope can be natural as identified, or further modified to better fit a specific HLA type. In some embodiments, neoepitopes can be scored / ranked based on multiplying the allele frequency by transcripts per million to obtain a likelihood score. This score can then be further enhanced using HLA information and the calculated or actual binding affinity for the patient's HLA type.

[0218] Among the embodiments provided are those in which neoepitopes are compared to a database containing known human sequences to avoid the use of human-identical sequences.

[0219] After in silico identification of suitable neoepitope sequences, corresponding synthetic peptides are prepared in vitro (e.g., using solid-phase synthesis). In certain embodiments, a library of synthetic peptides representing multiple different neoepitopes from a subject is prepared. The library can contain 100, 1000, 10,000, or more different peptides. It is contemplated that the in silico identified ones can be prepared in vitro to obtain synthetic peptides to obtain synthetic antibodies against the identified neoepitopes.

[0220] Synthetic peptides can be prepared using a variety of methods. For example, peptides containing cancer neoepitope sequences can be prepared using solid phase (e.g., using Merrified synthesis), via solution phase synthesis, or from relatively small peptide fragments. Peptide epitopes can be obtained by chemical synthesis using commercially available automated peptide synthesizers. In some embodiments, peptides can be synthesized using the Fmoc-polyamide mode of solid phase peptide synthesis, as disclosed, for example, by Lu et al. (1981). J. Org. Chem. 46, 3433 and references therein. In some aspects, peptides can be produced by expression of recombinant nucleic acids in a suitable host using a suitable expression system. In some aspects, recombinant methods can be used in which multiple neoepitopes are present on a single peptide chain, for example, with spacers between neoepitopes or cleavage sites.

[0221] Peptides can be purified by any one or a combination of techniques, such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and reversed-phase high performance liquid chromatography using, for example, acetonitrile / water gradient separation. In some embodiments, peptides can be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Peptide analysis can be performed using thin-layer chromatography, electrophoresis, particularly capillary electrophoresis, solid-phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.

[0222] B. Cell samples containing tumor-reactive T cells The provided methods include obtaining and enriching or selecting a T cell population from a biological sample for use as a first T cell population, or input population of T cells. In some cases, the first T cell population is known to contain T cells reactive to a tumor antigen, is likely to contain T cells reactive to a tumor antigen, or is capable of being reactive to a tumor antigen after ex vivo co-culture with, for example, an autologous source of tumor antigen. For example, typically, the first T cell population is derived from a biological sample from a tumor or from a subject known to have or likely to have a tumor. In certain embodiments, the first T cell population is further stimulated with one or more T cell stimulators (e.g., one or more recombinant cytokines, such as IL-2) and, optionally, one or more T cell adjuvants to generate a second or stimulated population of T cells containing expanded T cells.

[0223] In some embodiments, the conditions for stimulating T cells by culture with one or more T cell stimulators and, optionally, one or more T cell adjuvants, result in the expansion or growth of T cells present in the first T cell population, or the input population of T cells. In some embodiments, the conditions for stimulating T cells with one or more T cell stimulators and, optionally, one or more T cell adjuvants, can include culturing T cells under conditions that result in bulk expansion of T cells. In other particular embodiments, the conditions for stimulating T cells can include culturing T cells under conditions that result in preferential or favorable enrichment or growth of desired T cells while minimizing or reducing certain T cell subsets that may be undesirable.

[0224] In the provided methods, the stimulated T cell composition is then used in subsequent downstream steps for enrichment and expansion of tumor-reactive T cells, e.g., steps comprising co-culturing the stimulated T cells with antigen-presenting cells (APCs) in the presence of the T cell neoepitope (mutated) peptide antigen to generate, give rise to, or extract T cells that are tumor-reactive T cells. In certain embodiments, the provided methods can also include a step for selecting or enriching T cells reactive to tumor antigens (tumor-reactive T cells) after co-culturing the T cells with the APC / peptide neoepitope. The tumor-reactive T cell population can be cultured under expansion conditions, e.g., to generate a therapeutic T cell composition.

[0225] In some embodiments, the biological sample is from a subject with a tumor known to contain or likely to contain tumor-reactive T cells, and such T cells have been exposed to or activated by tumor neoantigens in vivo. In some embodiments, selecting T cells from the biological sample further comprises enriching or selecting tumor-reactive T cells or T cells that express one or more activation markers associated with tumor-reactive T cells. T cell activation markers include cell surface markers whose expression is upregulated or specific to T cells that have been exposed to an antigen and activated. Exemplary markers are described in Section ID below.

[0226] In any aspect of the provided methods, the input population of T cells, or the first population of T cells, is incubated in the presence of a T cell stimulator. In certain embodiments, the incubation is carried out under conditions where the T cell stimulator activates or stimulates the cells or promotes expansion of T cells present in the input population of T cells, or the first population of T cells.

[0227] In some embodiments, the T cell stimulator comprises a recombinant T cell stimulating cytokine, such as IL-2, IL-7, IL-15, and / or IL-21. In some embodiments, the T cell stimulating cytokine comprises IL-2 alone or in combination with another cytokine from among IL-7, IL-15, and / or IL-21. In some embodiments, the T cell stimulating cytokine is IL-7 and IL-15.

[0228] In some embodiments, the T cell stimulatory agent can include one or more agents that interact with CD3 and costimulatory molecules, such as CD28. The T cell stimulatory agent can include an anti-CD3 antibody, such as OKT3, and an anti-CD28 agent (presented by APCs or as a soluble antibody). In embodiments, T cells selected from a biological sample (i.e., input population or first population) are incubated in the presence of a T cell stimulatory agent, such as an anti-CD3 (e.g., OKT3) / anti-CD28 antibody, before and / or during at least a portion of the co-culture of T cells with APCs. Thus, either before co-culture in the presence of APCs or after selection of reactive cells, T cells are incubated with one or more T cell stimulatory agents of lymphocytes, such as, but not limited to, an anti-CD3 antibody (e.g., OKT3) and anti-CD28 (presented by APCs or as a soluble antibody), to generate a second T cell population comprising activated or stimulated T cells. In certain embodiments, one or more recombinant cytokines are also present during the incubation as additional T cell stimulants.

[0229] In some embodiments, incubation with a T cell stimulator is performed directly on an input population of T cells selected from a biological sample from a subject, or on a first T cell population, where the T cell population selected from the biological sample (e.g., autologous T cells from the subject) is incubated with the T cell stimulator. In other embodiments, the input population of T cells includes T cells that are likely or suspected to be tumor-reactive T cells, and such cells are first selected from a T cell population selected from a biological sample from a subject by selecting cells positive for a surface marker upregulated on activated T cells (e.g., 4-1BB or OX40). In such embodiments, incubation with a T cell stimulator is performed after enriching the T cell population for tumor-reactive T cells. In provided embodiments, incubation with a T cell stimulator is performed prior to co-culture of such T cells (stimulated T cells) with APC / peptide neoepitopes.

[0230] 1. Selection of T cell populations The provided methods include selecting or obtaining a population of T cells from a biological sample that can be used as a source or input of T cells for stimulation with one or more T cell stimulators (e.g., recombinant IL-1 and / or anti-CD3 / anti-CD28) and, in provided embodiments, a T cell modulator (e.g., a T cell agonist or an apoptosis inhibitor). In some embodiments, the T cells are derived from a biological sample from a subject known to contain or likely to contain tumor-reactive T cells. The collected biological sample contains or is suspected to contain lymphocytes with endogenous TCRs that are reactive to mutations present on the tumor.

[0231] In some aspects of the provided embodiments, a suitable biological sample is obtained from a subject, e.g., a patient of interest, i.e., a patient suspected or known to have cancer. In some embodiments, the sample is a sample known or suspected to contain T cells, e.g., T cells that are specific for, bind to, or likely to express an endogenous T cell receptor (TCR) that recognizes a tumor-associated antigen. The sample can be derived from any initial source that may contain or is suspected to contain such T cells. In some aspects, the source of the biological sample of interest includes, but is not limited to, many different physiological sources, such as tissue-derived samples, e.g., homogenates, and blood or derivatives thereof.

[0232] Any of a variety of samples can be used as a source of potentially reactive T cells. Tumors and downstream lymph nodes may have the highest frequency of reactive T cells (Powell et al., Clin. Cancer. Res., 2014), but other sample sources can also be used. In some cases, the sample is a tumor sample, a tertiary lymphatic site, a draining lymph node, peripheral blood, or bone marrow. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a lymph sample. In some embodiments, the sample is a peripheral blood sample.

[0233] Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, e.g., selection or enrichment, centrifugation, washing, and / or incubation. Biological samples can be samples obtained directly from biological sources or samples that have been processed. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, and processed samples derived therefrom.

[0234] In some aspects, the sample is a blood or blood-derived sample, or an apheresis product or leukapheresis product, or is derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organ, and / or cells derived therefrom. In the context of cell therapy, e.g., adoptive cell therapy, samples include samples from autologous and allogeneic sources.

[0235] In many embodiments, the sample can be derived from a body fluid that is at least suspected to contain T cells of interest. In many embodiments, a suitable initial source for the sample is blood. In some embodiments, the biological sample is a blood-derived sample. The blood-derived sample can be derived from whole blood or a fraction thereof, such as serum, plasma, etc., and in many embodiments, the sample is derived from blood cells collected from whole blood. In some aspects, the sample source contains mononuclear cells. For example, the biological sample is or contains peripheral blood mononuclear cells (PBMCs), or is derived from PBMCs.

[0236] In some embodiments, the sample is a PBMC-derived sample, and the sample is generally a fluid PBMC-derived sample. Any convenient method for generating a fluid PBMC sample can be used. In many embodiments, the fluid PBMC-derived sample is prepared by separating PBMC from whole blood, that is, by collecting PBMC, for example, by centrifugation (for example, by Ficoll-Hypaque density gradient centrifugation, where representative protocols for such separation procedures are disclosed in WO98 / 15646 and U.S. Patent No. 5,985,565).

[0237] In some embodiments, the sample is a tumor sample, thereby providing a source of tumor-infiltrating lymphocytes (TILs). In some aspects, TILs are T cells that have left the subject's bloodstream and migrated into or infiltrated the tumor. In certain aspects, the TILs are reactive to tumor antigens.

[0238] Patient tumor samples can be obtained by any of a variety of methods to obtain a sample containing a mixture of tumor cells and TIL cells. In some embodiments, tumor samples are obtained by surgical resection. In some embodiments, tumor samples are obtained by needle biopsy. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be derived from liquid tumors, e.g., tumors derived from hematological malignancies. Solid tumors can be any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach (gastrointestinal), and skin (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In certain embodiments, the tumor is any of those described in Section IV. In some embodiments, the tumor sample is derived from the same tumor source used to identify neoantigens for preparing peptide neoepitopes.

[0239] In provided embodiments, the tumor sample obtained is less than 1 mm in size. 3 or about 1 mm 3 From, 8mm 3 or about 8 mm 3 , e.g., 1 mm 3 or about 1 mm 3 From, 6mm 3 or about 6 mm 3 , 1mm 3 or about 1 mm 3 From, 4mm 3 or about 4 mm 3 , 1mm 3 or about 1 mm 3 From, 2mm 3 or about 2 mm 3 In some embodiments, the tumor fragments are about 2-3 mm 3 In some embodiments, the tumor fragment is about 1-2 mm 3 In some embodiments, the tumor fragments are obtained by physical fragmentation, for example, by scraping. In some embodiments, the tumor fragments are obtained by sharp scraping.

[0240] In some of any of the provided embodiments, the obtained tumor sample is fragmented into pieces ranging from 1 mm or about 1 mm in diameter to 8 mm or about 8 mm in diameter, e.g., 1 mm or about 1 mm in diameter to 6 mm or about 6 mm in diameter, 1 mm or about 1 mm in diameter to 4 mm or about 4 mm in diameter, 1 mm or about 1 mm in diameter to 2 mm or about 2 mm in diameter. In some embodiments, the tumor fragments are about 2-3 mm in diameter. In some embodiments, the tumor fragments are about 1-2 mm in diameter. In some embodiments, the tumor fragments are obtained by physical fragmentation, e.g., by scraping. In some embodiments, the tumor fragments are obtained by sharp scraping.

[0241] In some embodiments, the tumor sample is cryopreserved prior to fragmentation. In some embodiments, the tumor fragments are cryopreserved.

[0242] In some embodiments, the resulting tumor fragments are placed in culture medium with appropriate nutrients to maintain T cell expansion under conditions for maintaining T cell expansion, such as any of the conditions described in subsection IB2 below, for T cell stimulation. In some embodiments, 1-500 tumor fragments (e.g., 1-8 mm in size each) are placed in an appropriate culture vessel under expansion conditions. In some embodiments, 10, 20, 30, 40, 50, or more fragments are cultured under expansion conditions. The culture vessel can be a microwell, flask, tube, bag, or other closed system device. In some embodiments, the culture vessel is a sealed vessel that provides a gas-permeable surface area, such as a gas-permeable flask. Exemplary culture vessels that provide a gas-permeable surface area include G-Rex plates or flasks. In some embodiments, one tumor fragment (approximately 1-8 mm in diameter) is placed in approximately 2 cm of the culture vessel. 2 The specific culture vessel can be selected based on the number of tumor fragments available and / or the desired yield of cells. The selection of the culture vessel (e.g., G-Rex) can be selected by linearly scaling the number of fragments seeded to the surface area of ​​the culture vessel. In some embodiments, the surface area of ​​the culture vessel is approximately 2 cm. 2(e.g., a G-Rex 24-well plate), and about one tumor fragment (about 1-8 mm in diameter) is placed in the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 10 cm 2 (e.g., G-Rex 10 or G-Rex 10M), and about 5 tumor fragments (each about 1-8 mm in diameter) are placed in the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 100 cm. 2 (e.g., G-Rex 100 M / 100M-CS), and about 50 tumor fragments (each about 1-8 mm in diameter) are placed in the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 500 cm. 2 (e.g., G-Rex 500 M / 500M-CS), in which approximately 250 tumor fragments (each approximately 1-8 mm in diameter) are placed in a culture vessel. In aspects of the provided methods, increasing the size of the culture vessel, and therefore the number of tumor fragments per vessel, compared to methods involving smaller culture vessels and / or fewer fragments per vessel can reduce variability, e.g., by pooling more fragments to minimize inter-tumor variability between fragments.

[0243] In some embodiments, the tumor fragments are placed in culture medium to stimulate the cells using any of the conditions described in subsection IB2 below. In some embodiments, the culture medium is serum-free medium containing recombinant cytokines derived from IL-2, IL-7, IL-15, and / or IL-21, e.g., recombinant IL-12 or recombinant IL-7 and IL-15. The concentration of the recombinant cytokines can include any of those described. In particular embodiments, the culture medium is serum-free medium containing recombinant IL-2, e.g., from 300 IU / mL or about 300 IU / mL to 1000 IU / mL or about 1000 IU / mL, e.g., 300 IU / mL or about 300 IU / mL. In some embodiments, the culture medium is serum-free medium containing an anti-CD3 antibody and / or a CD28 targeting agent (e.g., an anti-CD28 antibody) and one or more recombinant cytokines (e.g., IL-2). In some embodiments, the culture medium contains one or more additional T cell stimulatory agonists or apoptosis inhibitors, as described in Section II.

[0244] In some embodiments, the provided methods include obtaining cells from tumor fragments, for example, by enzymatic digestion of the tumor fragments to obtain TILs. The enzymatic digestion can be performed using a collagenase, such as type IV collagenase or type I / II collagenase. The enzyme, such as collagenase, can be present in the medium for enzymatic digestion at a concentration of 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL, for example, 1 mg / mL or about 1 mg / mL, 2 mg / mL or about 2 mg / mL, 3 mg / mL or about 3 mg / mL, 4 mg / mL or about 4 mg / mL, or 5 mg / mL or about 5 mg / mL, or any value between the above. In some embodiments, the enzymatic digestion is performed in a medium containing, for example, 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL of type IV collagenase. In some embodiments, enzymatic digestion is with a medium containing, for example, 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL of type I / II collagenase. In other embodiments, enzymes from the Miltenyi Human Tumor Dissociation Kit can be used (e.g., catalog 0.130-095-929; Miltenyi Biotec). The enzyme-containing enzyme medium can be a serum-free medium, such as any of those described. In certain embodiments, the enzyme medium contains collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g., GlutaMAX), 10 mg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase. In some embodiments, the enzyme medium includes serum-free medium (e.g., OpTmizer) containing 2 mM glutamate (e.g., GlutaMAX), 10 μg / mL gentamicin, immune cell serum replacement (e.g., CTS immune cell serum replacement), and 1.0 mg / mL to 5.0 mg / mL collagenase. In some embodiments, the collagenase is type IV collagenase. In some embodiments, the collagenase is type I / II collagenase.

[0245] The tumor fragments are then mechanically cut to dissociate the TILs, for example, using a tissue dissociator. Tumor digests can be generated by placing the tumor in enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, followed by repeated cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. If the cell suspension at the end of this process contains a large number of red blood cells or dead cells, these cells can be removed by density gradient separation using FICOLL. Alternative methods known in the art, such as those described in U.S. Patent Application Publication No. 2012 / 0244133, the disclosure of which is incorporated herein by reference, may also be used. Any of the aforementioned methods can be used in any of the embodiments described herein for obtaining TILs for use in the provided methods.

[0246] In some embodiments, digested cells from tumor fragments are placed in culture medium with appropriate nutrients for maintaining T cell expansion under conditions for maintaining T cell expansion, such as any of the conditions described in subsection IB2 below, for stimulation of T cells. Cells are seeded at a specific density appropriate for the particular culture vessel. The culture vessel can be a microwell, flask, tube, bag, or other closed system device. In some embodiments, the culture vessel is a sealed vessel that provides a gas permeable surface area, such as a gas permeable flask. Exemplary culture vessels that provide a gas permeable surface area include G-Rex plates or flasks. In some embodiments, cells are seeded at a density of approximately 5 x 10 5 ~2×10 6 The enzymatically digested single cell suspension of 100 cells was placed in approximately 2 cm of the culture vessel. 2 The specific culture vessel can be selected based on the number of cells available and / or the desired yield of cells. The selection of the culture vessel (e.g., G-Rex) can be selected by linearly scaling the number of cells seeded to the surface area of ​​the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 2 cm. 2 (e.g., G-Rex 24-well plate), and approximately 5 × 105 ~2×10 6 The enzymatically digested single cell suspension of cells is placed in a culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 10 cm. 2 (e.g., G-Rex 10 or G-Rex 10M), which is approximately 2.5 × 10 6 ~1×10 7 The enzymatically digested single cell suspension of cells is placed in a culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 100 cm. 2 (e.g., G-Rex 100 M / 100M-CS), which is approximately 2.5 × 10 7 ~1×10 8 The enzymatically digested single cell suspension of cells is placed in a culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 500 cm. 2 (e.g., G-Rex 500 M / 500M-CS), which is approximately 1.25 × 10 8 ~5×10 8 An enzymatically digested single cell suspension of 100 cells is placed in a culture vessel.

[0247] In some embodiments, the culture medium is serum-free medium containing recombinant IL-2. In some embodiments, one or more additional T cell stimulatory agents can also be included. In some embodiments, the culture medium is serum-free medium containing an anti-CD3 antibody and / or a CD28 targeting agent (e.g., an anti-CD28 antibody) and one or more recombinant cytokines (e.g., IL-2). In some embodiments, the culture medium contains one or more additional T cell stimulatory agonists or apoptosis inhibitors, as described in Section II.

[0248] Samples can be obtained from a variety of different subjects / patients / hosts. Generally, such hosts are "mammals" or "mammals," which terms are used broadly to refer to organisms belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the host is human.

[0249] In some aspects, the subject is human.Thus, in some embodiments, the cell is a primary cell, for example, a primary human cell.In some embodiments, the sample is autologous to the subject to be treated, for example, the subject is a patient who needs a specific therapeutic intervention, such as adoptive cell therapy, in which cells are isolated, processed, and / or expanded according to the provided method.In some embodiments, the sample is allogeneic to the subject to be treated.

[0250] In some embodiments, T cells for use in connection with the provided methods can be enriched or sorted by a variety of methods, including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed-cartridge-based cell sorters. In certain aspects, one or more reagents specific for T cells or subsets thereof, such as reagents specific for T cell activation markers to select reactive cells, can be used, including, but not limited to, fluorescent antibodies, nanoparticles, or beads on cell sorting equipment, including, but not limited to, CliniMACS, Sony FX500, or Tyto cell sorting systems (Miltenyi).

[0251] In some aspects, T cells can be selected from a biological sample based on, for example, T cell markers CD3, CD4, or CD8. In some embodiments, selecting T cells that are surface positive for one or more cell surface markers includes any method for separation based on such markers.

[0252] In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some aspects, the isolation involves separating cells and cell populations based on the cellular expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or a binding partner that specifically binds to such markers, typically followed by a washing step and separating the cells bound to the antibody or binding partner from those not bound to the antibody or binding partner. In some embodiments, immunoaffinity-based selection involves contacting a sample containing cells, such as a sample containing CD3+ T cells, or a bulk population of T cells, e.g., primary human T cells, with an antibody or binding partner that specifically binds to one or more cell surface markers. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as spheres or beads, e.g., nanoparticles, microbeads, nanobeads including agarose, magnetic beads, or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection. In some embodiments, the spheres or beads can be packed into a column to perform immunoaffinity chromatography, in which a sample containing cells, such as CD3+ T cells or primary human T cells, including CD4+ and CD8+ cells, is contacted with the matrix of the column and subsequently eluted or released therefrom. In other embodiments, the antibody or binding partner is detectably labeled.

[0253] In some aspects, a sample or composition of cells to be separated is incubated with a small magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, e.g., nanoparticles or paramagnetic beads. The magnetically responsive material, e.g., particles, is generally attached directly or indirectly to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., a surface marker, present on one or more cells or cell populations desired to be separated, e.g., negatively or positively selected. Such beads are known and, in some aspects, are commercially available from a variety of sources, including Dynabeads® (Life Technologies, Carlsbad, CA), MACS® beads (Miltenyi Biotec, San Diego, CA), or Streptamer® bead reagents (IBA, Germany). In some aspects, the sample is placed in a magnetic field, and cells with attached magnetically responsive or magnetizable particles are attracted to the magnet and separated from unlabeled cells. In the case of positive selection, cells attracted to the magnet are retained. In the case of negative selection, non-attracted cells (unlabeled cells) are retained.

[0254] In certain embodiments, the sample is contacted with a binder that specifically binds to the cell surface marker, e.g., a detectably labeled binder. In certain embodiments, the detectably labeled binder is fluorescently labeled. In certain embodiments, T cells labeled by the binder specific for the cell surface marker are identified by flow cytometry. In certain embodiments, the method further includes separating any resulting T cells labeled by the binder from other components of the sample to produce a composition enriched for T cells that are surface-positive for one or more cell surface markers. Cell sorting equipment with a sufficiently high throughput to handle large volumes and cell numbers can be used. Non-limiting examples of cell sorting equipment include, for example, the Sony FX500 or the Tyto cell sorting system (Miltenyi).

[0255] Incubation is generally carried out under conditions in which the antibody or binding partner, or a molecule, e.g., a secondary antibody or other reagent that specifically binds to such an antibody or binding partner attached to a magnetic particle or bead and / or detectably labeled, specifically binds to a cell surface molecule if present on cells in the sample. In some aspects, antibody-bound cells can be recovered or separated from unbound cells in the sample.

[0256] In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subjected to additional separation steps. Such separation steps can be based on positive selection, in which cells bound to the reagent are retained for subsequent use, and / or negative selection, in which cells that are not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for subsequent use. In some aspects, negative selection, which specifically identifies cell types within a heterogeneous population, can be particularly useful when antibodies are not available, so that separation is best performed based on markers expressed by cells other than the desired population.

[0257] Separation does not necessarily result in the enrichment or removal of 100% of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, indicates an increase in the number or proportion of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, indicates a decrease in the number or proportion of such cells, but does not necessarily result in the complete removal of any such cells. For example, in some aspects, the selection of one of the CD4+ or CD8+ populations enriches the population, either the CD4+ or CD8+ population, but may also contain some remaining unselected cells, or a small percentage of other unselected cells, which may include the other of the CD4 or CD8 population, still present in the enriched population.

[0258] In some embodiments, isolation is achieved by enriching for a particular cell population by positive selection, or by depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by determining whether a specific cell population is expressed or at relatively high levels (markers) in the positively or negatively selected cells, respectively. high ) expressed (marker + This is accomplished by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers selected.

[0259] In certain embodiments, the T cell population comprises both CD4+ T cells and CD8+ T cells. In some cases, the CD4+ cell population and the CD8+ T cell population are isolated, selected or enriched from biological samples. Many cancers, including solid tumors, for example, many common epithelial indications (e.g., GI), express class I restriction mutations and class II restriction mutations. For T cell products to target such indications, for example, common epithelial indications, it is believed that both CD8+ T cells recognize class I MHC restriction molecules and CD4+ T cells recognize class II MHC restriction molecules.

[0260] In some embodiments, the methods involve the isolation, selection and / or enrichment of CD3+ cells. In some embodiments, the methods involve the isolation, selection and / or enrichment of CD4+ cells and CD8+ cells. In some aspects, CD4 + or CD8 + a selection step, e.g., using positive selection for CD4 and positive selection for CD8, + Helper T cells and CD8 +In some aspects, such selection is performed simultaneously, and in other aspects, sequentially in either order. In some embodiments, the method includes enriching for CD4+ T cells and CD8+ T cells by selecting for CD3 surface-positive T cells, or by sequentially or simultaneously selecting for CD4 surface-positive T cells and CD8 surface-positive T cells. Such CD3+ T cells, or CD4 + and / or CD8 + The population can be further sorted into subpopulations by positive or negative selection for markers expressed or relatively highly expressed on tumor-reactive T cells, or T cells with expression of T cell activation markers associated with tumor-reactive T cells, for example, as described in Section ID.

[0261] In some embodiments, the isolation of a cell or population further comprises one or more preparative and / or affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, or lyse or remove cells sensitive to a particular reagent. In some examples, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity, and / or resistance to a particular component.

[0262] In some embodiments, the selected population is enriched for CD3+ T cells and comprises CD3+ T cells as a percentage of total cells in the population that is greater than or about 60%, greater than or about 70%, greater than or about 70%, greater than or about 80%, greater than or about 90%, or greater than or about 95%. In some embodiments, the selected population is enriched for CD4+ T cells and CD8+ T cells and comprises CD4+ T cells and CD8+ T cells as a percentage of total cells in the population that is greater than or about 60%, greater than or about 70%, greater than or about 70%, greater than or about 80%, greater than or about 90%, or greater than or about 95%. In specific embodiments, the ratio of CD8+ T cells to CD4+ T cells is from at or about 1:100, 100:1 or about 100:1, 1:50 or about 1:50, 50:1 or about 50:1, 1:25 or about 1:25, 25:1 or about 25:1, 1:10 or about 1:10, 10:1 or about 10:1, 1:5 or about 1:5, 5:1 or about 5:1, or 1:2.5 or about 1:2.5, 2.5:1 or about 2.5:1.

[0263] In some of any of the provided embodiments, the biological sample is a peripheral blood sample, optionally an apheresis sample, and the number of cells at the start of the culture is 1 x 10 9 pieces or approximately 1 x 10 9 From 7 x 10 9 total viable cells; or 1 x 10 9 or approximately 1 x 10 9 Total viable cells, 2 x 10 9 or approximately 2 x 10 9 Total viable cells, 3 x 10 9 Total viable cells, 4 x 10 9 Total viable cells, 5 x 10 9 Total viable cells, 6 x 10 9 total viable cells, or 7 x 10 9and / or the percentage of tumor-reactive T cells at the initiation of the culture is from 0.02% or about 0.02% to 40% or about 40%, from 0.02% or about 0.02% to 24% or about 24%, from 0.02% or about 0.02% to 18% or about 18%, from 0.02% or about 0.02% to 0.9% or about 0.9%, or from 0.02% or about 0.02% to 6.0% or about 6.0%; and / or the number of T cells surface-positive for a T cell activation marker at the initiation of the culture is 0.1 x 10 6 or approximately 0.1 x 10 6 From 60 x 10 6 pieces or approximately 60 x 10 6 T cells, 0.1 x 10 6 From 8 x 10 6 pieces or approximately 8 x 10 6 T cells, 0.1 x 10 6 From 20 x 10 6 pieces or approximately 20 x 10 6 T cells, 0.3 x 10 6 From 35 x 10 6 pieces or approximately 35 x 10 6 T cells, or 0.3 x 10 6 From 60 x 10 6 pieces or approximately 60 x 10 6 or 0.1 x 10 T cells 6 T cells, 0.3 x 10 6 T cells, 0.6 x 10 6 T cells, 1 x 10 6 T cells, 5 x 10 6 T cells, 10 x 10 6 T cells, 35 x 10 6 T cells, or 60 x 10 6 T cells or approximately 0.1 x 10 6 T cells, 0.3 x 10 6 T cells, 0.6 x 10 6 T cells, 1 x 10 6 T cells, 5 x 10 6 T cells, 10 x 10 6 T cells, 35 x 106 T cells, or 60 x 10 6 T cells, or any value between any of the aforementioned.

[0264] In some of any of the provided embodiments, the biological sample is a lymphoid-derived sample or a tumor-derived sample, and the number of cells at the start of the culture is 10 x 10 6 pieces or approximately 10 x 10 6 From 100 x 10 6 Total viable cells, 20 x 10 6 From 100 x 10 6 total viable cells, or 12 x 10 6 From 43 x 10 6 total viable cells; or 10 x 10 6 pieces or approximately 10 x 10 6 Total viable cells, 12 x 10 6 pieces or approximately 12 x 10 6 Total viable cells, 20 x 10 6 Total viable cells, 40 x 10 6 Total viable cells, 60 x 10 6 total viable cells, or 100 x 10 6 total viable cells, or any value between any of the foregoing; and / or the percentage of tumor-reactive T cells at the initiation of the culture is from 1% or about 1% to 90% or about 90%, from 1% or about 1% to 75% or about 75%, from 1% or about 1% to 50% or about 50%, from 1% or about 1% to 25% or about 25%, or from 1% or about 1% to 14% or about 14%; and / or the number of T cells surface-positive for a T cell activation marker at the initiation of the culture is 0.7 x 10 6 or approximately 0.7 x 10 6 From 15 x 10 6 pieces or approximately 15 x 10 6 T cells, 1 x 10 6 From 15 x 10 6 pieces or approximately 15 x 10 6 T cells, or 0.7 x 10 6 or approximately 0.7 x 10 6 From 5.4 x 10 6or approximately 5.4 x 10 6 or 0.7 x 10 T cells 6 T cells, 1 x 10 6 T cells, 5.4 x 10 6 T cells, or 15 x 10 6 T cells or approximately 0.7 x 10 6 T cells, 1 x 10 6 T cells, 5.4 x 10 6 T cells, or 15 x 10 6 T cells, or any value between any of the aforementioned.

[0265] In some embodiments, the selected T cells can be further enriched for tumor-reactive T cells based on the expression of markers associated with activated T cells. Specific markers for use in selecting or enriching such tumor-reactive T cells are described in Section ID below. In other cases, selection or enrichment of tumor-reactive T cells occurs at one or more subsequent stages of the process, such as after co-culture with one or more mutant peptides (peptide neoepitopes).

[0266] 2. Stimulation of T cells for initial expansion In aspects of the provided methods, T cells from a biological sample (e.g., an input population of T cells present in a resected tumor fragment, or a first T cell population) are incubated or cultured under conditions to stimulate the T cells, e.g., in the presence of one or more T cell stimulators, to expand the T cells. Optionally, the culturing or incubation is further carried out in the presence of one or more T cell modulators or adjuvants, e.g., T cell agonists or apoptosis inhibitors. In some embodiments, incubation or culture with one or more T cell stimulators and / or T cell modulators or adjuvants results in the expansion or growth of selected T cells, or a desired subset or subtype thereof, or live cells thereof, for use in subsequent steps of the provided methods. Non-limiting examples of T cell stimulators and / or T cell modulators or adjuvants and conditions for incubation or culture are described herein.

[0267] In some embodiments, the T cell stimulator comprises a recombinant T cell stimulating cytokine, such as IL-2, IL-7, IL-15, and / or IL-21. In some embodiments, the T cell stimulating cytokine comprises IL-2 alone or in combination with another cytokine from among IL-7, IL-15, and / or IL-21. In some embodiments, the T cell stimulating cytokine comprises IL-7 and IL-15.

[0268] In any aspect of the provided methods, the population of T cells is incubated in the presence of a T cell stimulator. In certain embodiments, the incubation is under conditions in which the T cell stimulator activates or stimulates the cells or promotes expansion of the cells.

[0269] In some of the provided embodiments, the T cell stimulatory agent is selected from an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling through a costimulatory receptor. In some of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, such as OKT3. In some of the provided embodiments, the agent that initiates signaling through a costimulatory receptor comprises peripheral blood mononuclear cells (PBMCs), optionally non-dividing PBMCs or irradiated PBMCs. In some of the provided embodiments, the agent that initiates signaling through a costimulatory receptor is an anti-CD28 antibody. In some of the provided embodiments, the T cell stimulatory agent is a soluble anti-CD3 antibody and an anti-CD28 antibody, respectively.

[0270] Thus, among the methods provided are methods of culturing T cells to produce tumor-reactive T cells, in which the T cells are cultured or incubated in the presence of a T cell stimulator under conditions that expand the T cells as present in the co-culture. In some embodiments, the T cell stimulator is or includes an anti-CD3 antibody and an anti-CD28 antibody.

[0271] In embodiments of the provided methods, the stimulatory conditions include one or more agents, e.g., ligands, that turn on or initiate the TCR / CD3 intracellular signaling cascade in the T cell and / or a costimulatory signal in the T cell. Such agents can include antibodies, e.g., those specific for TCR components, e.g., anti-CD3 and / or costimulatory receptors, e.g., anti-CD28 or anti-4-1BB. In some embodiments, such agents are added to the culture medium as soluble antibodies. In other embodiments, such agents are bound to a solid support, such as beads. In some embodiments, the T cell stimulatory agent comprises anti-CD3 / CD28-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0272] Anti-CD3 antibodies can include any antibody that is directed against or can specifically bind to the CD3 receptor on the surface of T cells, typically human CD3 on human T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include UHCTI clones, also known as T3 and CD3E. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. Anti-CD3 antibodies can be added as a soluble reagent or can be bound to beads. In certain embodiments, the anti-CD3 antibody is soluble.

[0273] In certain embodiments, the T cell stimulator comprises an anti-CD3 antibody that is added t...

Claims

1. A method for producing tumor-reactive T cells, comprising: (1) (a) incubating a cell population comprising T cells from a tumor sample obtained from a subject with a first T cell stimulator comprising one or more recombinant cytokines selected from IL-2, IL-7, IL-15, and IL-21 under conditions to stimulate expansion of T cells in the population to generate a stimulated T cell population; (b) co-culturing the stimulated T cell population in the presence of antigen-presenting cells (APCs) that present one or more peptide neo-antigens derived from a tumor from the subject on a major histocompatibility complex (MHC), thereby generating a population containing T cells, including tumor-reactive T cells; (c) enriching a population of tumor-reactive T cells from the co-culture that are reactive to the one or more peptide neo-antigens, thereby generating a T cell population enriched for tumor-reactive T cells, wherein the tumor-reactive T cells comprise endogenous T cell receptors (TCRs) that are reactive to the one or more peptide neo-antigens presented on the APCs, and wherein enriching the tumor-reactive T cells comprises selecting T cells that are surface-positive for one or more T cell activation markers that are CD134 and / or CD137; and (d) incubating the T cell population enriched for tumor-reactive T cells with a second T cell stimulator comprising one or more recombinant cytokines selected from IL-2, IL-7, IL-15, and IL-21 under conditions to stimulate expansion of T cells in the population. Culturing the T cells by a process comprising: (2) harvesting the cells produced by the method to produce a composition of expanded T cells enriched for tumor-reactive T cells; Including, A method wherein one or more steps of the culturing step are performed in the presence of at least one T cell adjuvant that is (i) an apoptosis inhibitor that inhibits caspase activation or activity, (ii) a costimulatory agonist, or (iii) a checkpoint inhibitor.

2. The apoptosis inhibitor is (i) a concentration of about 0.5 μM to about 50 μM, inclusive; or (ii) a concentration of about 0.5 μg / mL to 25 μg / mL or about 25 μg / mL, inclusive; 2. The method of claim 1, wherein

3. the costimulatory agonist is a tumor necrosis factor receptor superfamily (TNFRSF) agonist; and / or the costimulatory agonist is an antibody or antigen-binding fragment that specifically binds to a TNFRSF member, or a fusion protein that includes the extracellular domain of a ligand of the TNFRSF member or a binding portion thereof; 3. The method of claim 1 or 2.

4. The method of claim 3, wherein the TNFRSF member is selected from OX40, 4-1BB, GITR, and CD27, and / or the costimulatory agonist specifically binds to OX40, 4-1BB, GITR, or CD27.

5. The method of any one of claims 1 to 4, wherein the checkpoint inhibitor inhibits the activity of an immune checkpoint protein selected from the group consisting of PD-1 / PD-L1, CTLA-4, OX40, LAG-3, TIM-3, and B7-H3.

6. the checkpoint inhibitor (i) anti-PD-1 antibody, (ii) anti-PDL1 antibody, (iii) anti-OX40L antibody, and / or (iv) ipilimumab or an antigen-binding fragment thereof The method according to any one of claims 1 to 5, wherein

7. The method of any one of claims 1 to 6, wherein the first T cell stimulator comprises recombinant IL-2.

8. The method of any one of claims 1 to 7, wherein the second T cell stimulator comprises recombinant IL-2.

9. 9. The method of any one of claims 1 to 8, wherein the concentration of each of said one or more recombinant cytokines individually is between 100 IU / mL and 6000 IU / mL.

10. 10. The method of any one of claims 1 to 9, wherein the concentration of each of said one or more recombinant cytokines individually is 300 IU / mL to 6000 IU / mL, 300 IU / mL to 3000 IU / mL, or 300 IU / mL to 1000 IU / mL.

11. 11. The method of any one of claims 1 to 10, wherein the concentration of each of said one or more recombinant cytokines individually is at or about 300 IU / mL, 1000 IU / mL or about 1000 IU / mL, or 6000 IU / mL or about 6000 IU / mL.

12. The method of any one of claims 1 to 11, wherein the second T cell stimulator further comprises an anti-CD3 antibody.

13. The method of claim 12, wherein the anti-CD3 antibody is OKT3.

14. 14. The method of any one of claims 1 to 13, wherein the second T cell stimulator comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMCs) and / or feeder cells that are gamma-irradiated non-dividing peripheral blood mononuclear cells (PBMCs).

15. The method of any one of claims 1 to 14, wherein the antigen-presenting cell is a nucleated cell, such as a dendritic cell, a mononuclear phagocyte, a B lymphocyte, an endothelial cell, or a thymic epithelium.

16. The method of any one of claims 1 to 15, wherein the antigen-presenting cells are dendritic cells.

17. The method of any one of claims 1 to 16, wherein the antigen-presenting cells are autologous to the subject.

18. 18. The method of any one of claims 1 to 17, wherein said one or more peptide neoantigens comprise at least one neoepitope from said subject.

19. 19. The method of any one of claims 1 to 18, wherein said one or more peptide neoantigens comprise individual peptides or peptide pools.

20. The method of any one of claims 1 to 19, wherein the MHC molecule is a class I molecule, a class II molecule, or an MHC class I molecule and an MHC class II molecule.

21. 21. The method of any one of claims 1 to 20, wherein the T cells are CD4+ cells, CD8+ cells, or CD4+ cells and CD8+ cells.

22. The method of any one of claims 1 to 21, wherein the co-culture ratio of antigen-presenting cells to T cells is between 20:1 and 1:1, and / or the co-culture ratio of antigen-presenting cells to T cells is 1:1 or approximately 1:

1.

23. The method of any one of claims 1 to 22, wherein the co-culture ratio of antigen-presenting cells to T cells is 1:1 or about 1:

1.

24. The method of any one of claims 1 to 23, wherein said co-cultivation lasts for 2 hours to 24 hours.

25. The method of any one of claims 1 to 24, wherein said co-cultivation is for 6 hours or about 6 hours.

26. The method of any one of claims 1 to 25, wherein the one or more T cell activation markers are CD134 and CD137.

27. 27. The method of any one of claims 1 to 26, wherein one or more of the method steps are carried out in a closed system.

28. 28. The method of any one of claims 1 to 27, wherein said incubating with the first T cell stimulator is for 7 to 21 days.

29. 29. The method of any one of claims 1 to 28, wherein said incubating with said second T cell stimulator is for 7 to 21 days.

30. 30. The method of any one of claims 1 to 29, wherein the method comprises formulating the harvested cells with a cryoprotectant.

31. 31. The method of any one of claims 1 to 30, wherein selecting T cells surface positive for the one or more T cell activation markers is by flow cytometry.

32. The method of any one of claims 1 to 31, wherein the tumor is an epithelial cancer tumor.

33. 32. The method of any one of claims 1 to 31, wherein the tumor is a melanoma, lung squamous cell, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, gastric cancer, or uterine cancer tumor.

34. 32. The method of any one of claims 1 to 31, wherein the tumor is a non-small cell lung cancer (NSCLC), ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer tumor.

35. The method of any one of claims 1 to 31, wherein the tumor is a cutaneous melanoma tumor.

36. Expanded T cells enriched for tumor-reactive T cells produced by the method of any one of claims 1 to 35. A composition comprising:

37. 37. The composition of claim 36, for use in autologous therapy for treating cancer in a subject.

38. 37. Use of the composition of claim 36 in the manufacture of a medicament for autotherapy to treat cancer in a subject.

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