EX-VIVO METHOD FOR THE PRODUCTION OF A T-CELL THERAPY AND ASSOCIATED COMPOSITIONS AND METHOD

Description

Field

[0001] The present disclosure provides methods for ex vivo expansion of a T cells, including tumor-reactive T cells, and compositions containing such T cells for use in methods for treating cancer.Background

[0002] Cancer cells accumulate lots of different DNA mutations as part of the tumorigenic process. These mutations can cause amino acid changes in protein coding regions. For a mutation to be recognized by the immune system the protein needs to be processed intracellularly and present the mutant peptide presented on the surface with the Major Histocompatibility Complex (MHC). Neoantigens are the mutant peptides presented by the MHC complex that can be recognized by a T-cell via TCR binding. Neoantigens are ideal targets for immunotherapies. These antigens were not present in the body before the cancer developed and are truly cancer specific, not expressed on normal cells and are not subjected to off target immune toxicity. Clinical studies have demonstrated that T cells isolated from surgically resected tumor possess TCRs that recognize neoantigens, and expanding these neoantigen reactive TIL populations and re-infusing them into the patient can in some cases result in a dramatic clinical benefit. However, a major obstacle to applications of such cells in cell therapy is the difficulty in obtaining such cells. Improved methods are needed for obtaining and manufacturing cell compositions containing tumor-reactive T cells for therapeutic use. Provided herein are embodiments that meet such needs. Yossef et al. JCI Insight, 3 (2018) describes a method of enhanced detection of neoantigen-reactive T cells targeting unique and shared oncogenes for personalized cancer immunotherapy.Summary of the invention

[0003] The present invention, as defined in the claims, provides a method for manufacturing tumor-reactive T cells, the method comprising: (1) culturing T cells by a process that comprises: (a) incubating a population of cells comprising T cells from a biological sample obtained from a subject that has a tumor with a first T cell stimulatory agent(s) under conditions to stimulate expansion of T cells of the population to produce a population of stimulated T cells, wherein the first T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines; (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (c) enriching from the co-culture the population of tumor reactive T cells reactive to the one or more peptides, wherein said tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and (d) incubating the population of T cells enriched in tumor reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of T cells in the population, wherein the second T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines; and wherein one or more steps of the culturing is carried out 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 which is a tumor necrosis factor receptor superfamily (TNFRSF) agonist, or (iii) a checkpoint inhibitor which 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; and (2) harvesting cells produced by the method to produce a composition of expanded T cells enriched in tumor reactive T cells.

[0004] The invention also provides a composition comprising expanded T cells enriched in tumor reactive T cells for use in a method of treating a subject having a cancer, wherein the method comprises manufacturing the composition using the method of any one of the claims and administering the composition to a subject having a tumor a therapeutic dose of the composition.

[0005] Further aspects of the invention and preferred embodiments are defined in the dependent claims. Any aspects, embodiments and examples of the present disclosure which do not fall under the scope of the appended claims do not form part of the invention and are merely provided for illustrative purposes.Summary of the technical teachings

[0006] The technical information set out below may in some respects go beyond the disclosure of the invention, which is defined by the appended claims. The additional technical information is provided to place the actual invention in a broader technical context and to illustrate possible related technical developments. Such additional technical information which does not fall within the scope of the appended claims, is not part of the invention. Accordingly, any aspects, embodiments and examples of the present disclosure which do not fall under the scope of the appended claims do not form part of the invention and are merely provided for illustrative purposes.

[0007] Provided herein is a method for manufacturing tumor-reactive T cells, the method comprising culturing T cells by a process that comprises: (a) incubating a population of cells comprising T cells from a biological sample obtained from a subject with a T cell stimulatory agent(s) under conditions to stimulate expansion of T cells of the population to produce a population of stimulated T cells; (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (c) enriching from the co-culture the population of tumor reactive T cells reactive to the one or more peptides; and (d) incubating the population of T cells enriched in tumor reactive T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of T cells in the population, wherein one or more steps of the culturing is carried out in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor. In some embodiments, the method further comprises (e) harvesting cells produced by the method.

[0008] Provided herein is a method for manufacturing tumor-reactive T cells, the method comprising (1) culturing T cells by a process that comprises: (a) incubating a population of cells comprising T cells from a biological sample obtained from a subject that has a tumor with a first T cell stimulatory agent(s) under conditions to stimulate expansion of T cells of the population to produce a population of stimulated T cells; (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (d) enriching from the co-culture the population of tumor reactive T cells reactive to the one or more peptides, wherein said tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and (e) incubating the population of T cells enriched in tumor reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of T cells in the population; and wherein one or more steps of the culturing is carried out in the presence of at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis that is an apoptosisinhibitor that inhibits caspase activation or activity; and (2) harvesting cells produced by the method to produce a composition of expanded T cells enriched in tumor reactive T cells.

[0009] In some of any of the provided embodiments, the first or second T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines, wherein the incubating in the presence of the T cell stimulatory agent(s) is carried out prior to, during and / or subsequent to the incubating in the presence of the one or more T cell adjuvants.

[0010] In some of any of the provided embodiments, the incubation with the at least one T cell adjuvant is carried out during at least a portion of the co-culturing. In some of any of the embodiments, at least a portion of the incubating the population of T cells with the at least one T cell adjuvant is carried out concurrently with the incubating the population of T cells with the T cell stimulatory agent(s).

[0011] Provided herein is a method for manufacturing tumor-reactice T cells, the method comprising: (1) culturing T cells by a process comprising: (a) incubating a population of cells comprising T cells from a biological sample from a subject that has a tumor with a first T cell stimulatory agent(s) comprising one or more recombinant cytokinesunder conditions to stimulate expansion of T cells in the population to produce a population of stimulated T cells, wherein the incubation with the one or more recombinant cytokines is in the presence of at least one T cell adjuvantthat is an apoptosis inhibitor that inhibits caspase activation or activity; (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (c) enriching tumor-reactive T cells from the co-culture, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (d) incubating the population of T cells enriched for tumor-reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells; and (2) harvesting cells produced by the method.

[0012] In some of any of the provided embodiments, the at least one T cell adjuvant further comprises at least one costimulatory agonist. In some of any of the provided embodiments, the incubating with the at least one costimulatory agonist and the apoptosis inhibitor is carried out simultaneously, intermittenttly or sequentially.

[0013] 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 is carried out simultaneously, intermittenttly or sequentially.

[0014] Provided herein is a method for manufacturing tumor-reactive T cells, the method comprising: (1) culturing T cells by a process that comprises: (a) incubating a population of cells comprising T cells from a biological sample obtained from a subject that has a tumor with a first T cell stimulatory agent(s) under conditions to stimulate expansion of T cells of the population to produce a population of stimulated T cells; (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (d) enriching from the co-culture the population of tumor reactive T cells reactive to the one or more peptides, wherein said tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and (d) incubating the population of T cells enriched in tumor reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of T cells in the population; and wherein one or more steps of the culturing is carried out in the presence of at least one T cell adjuvant selected from that is a costimulatory agonist or an apoptosis; and (2) harvesting cells produced by the method to produce a composition of expanded T cells enriched in tumor reactive T cells.

[0015] In some of any of the provided embodiments, the first or second T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines, wherein the incubating in the presence of the T cell stimulatory agent(s) is carried out prior to, during and / or subsequent to the incubating in the presence of the one or more T cell adjuvants.

[0016] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising (1) culturing T cells by a process comprising incubating an ex vivo population of T cells comprising tumor-reactive T cells, said tumor-reactive T cells comprising an endogenous TCR that is 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 population of T cells is enriched for CD4+ and CD8+ T cells and wherein at least a portion of the incubation is carried out prior to, concurrently with, or subsequent to incubating T cells with a T cell stimulatory agent(s) under conditions to activate T cells, wherein the culturing stimulates expansion of cells of the population of T cells; and (2) harvesting cells produced by the method.

[0017] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) incubating a population of cells comprising T cells from a biological sample from a subject that has a tumor with a first T cell stimulatory agent(s) comprising one or more recombinant cytokinesunder conditions to stimulate expansion of T cells in the population to produce a population of stimulated T cells, 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 population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (c) enriching tumor-reactive T cells from the co-culture, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (d) incubating the population of T cells enriched for tumor-reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells; and (2) harvesting the expanded cells produced by the method.

[0018] In some of any of the provided embodiments, at least one T cell adjuvant further comprises at least one apoptosis inhibitor. In some of any of the provided embodiments, at least one apoptosis inhibitor and the checkpoint agonist is carried out simultaneously, intermittently or sequentially. In some of any of the provided embodiments, at least one T cell adjuvant further comprises at least one checkpoint inhibitor. In some of any of the embodiments, the at least one checkpoint inhibitor and the costimulatory agonist is carried out simultaneously, intermittently or sequentially.

[0019] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) incubating an ex vivo population of T cells comprising tumor-reactive T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, wherein the population of T cells is enriched for CD4+ and CD8+ T cells; and (b) incubating T cells with a T cell stimulatory agent(s) under conditions to activate T cells, wherein at least a portion of the incubating is carried out prior to, concurrently with, or subsequent to the incubating in (a), wherein the culturing stimulates expansion of cells of the population of T cells; and (2) harvesting cells produced by the method.

[0020] Provided herein is a method for manufacturing tumor-reactive T cells, the method comprising: (1) culturing T cells by a process that comprises: (a) incubating a population of cells comprising T cells from a biological sample obtained from a subject that has a tumor with a first T cell stimulatory agent(s) under conditions to stimulate expansion of T cells of the population to produce a population of stimulated T cells: (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (d) enriching from the co-culture the population of tumor reactive T cells reactive to the one or more peptides, wherein said tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and (d) incubating the population of T cells enriched in tumor reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of T cells in the population; and wherein one or more steps of the culturing is carried out in the presence of at least one T cell adjuvant-that is a checkpoint inhibitor; and (2) harvesting the expanded cells produced by the method.

[0021] In any of the embodiments, the culturing the T cells further comprises incubating the cells in the presence of one or more recombinant cytokines, wherein the incubating in the presence of one or more cytokines is carried out prior to, during and / or subsequent to the incubating with the T cell stimulatory agents(s) and / or incubating in the presence of the one or more T cell adjuvants.

[0022] In some of any of the provided embodiments, the first or second T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines, wherein the incubating in the presence of the T cell stimulatory agent(s) is carried out prior to, during and / or subsequent to the incubating in the presence of the one or more T cell adjuvants. In some of any of the provided embodiments, the incubation with the at least one T cell adjuvant is carried out during at least a portion of the co-culturing. In some of any of the provided embodiments, at least a portion of the incubating the population of T cells with the at least one T cell adjuvant is carried out concurrently with the incubating the population of T cells with the T cell stimulatory agent(s).

[0023] In some of any of the embodiments, the T cell stimulating agent(s) comprises the presence of one or more recombinant cytokines, wherein the incubating in the presence of the T cell stimulatory agent(s) is carried out prior to, during and / or subsequent to the incubating in the presence of the one or more T cell adjuvants.

[0024] In some of any of the embodiments, the T cell stimulating agent(s) comprises the an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling via a costimulatory receptor, optionally wherein the costimulatory receptor is CD28, wherein the incubating in the presence of the T cell stimulating agent(s) is carried out prior to, during and / or subsequent to the incubating in the presence of the one or more T cell adjuvants.

[0025] In some of any of the provided embodiments, the method further comprises washing the cells during one or more of the steps for culturing, such as during incubation with T cell stimulatory agent(s) and / or the one or more T cell adjuvants, e.g. costimulatory agonist, apoptosis inhibitor (e.g. caspase inhibitor), or checkpoint inhibitor. In some embodiments, the provided methods include adding during the one or more steps a transient addition (e.g. only one time, such as at the initiation of the incubation or culture) of the T cell adjuvant, e.g. costimulatory agonist, apoptosis inhibitor (e.g. caspase inhibitor), or checkpoint inhibitor. In some embodiments, the provided methods include continuous addition of the T cell adjuvant during the one or more steps. For example, the one or more T cell adjuvants, e.g. costimulatory agonist, apoptosis inhibitor (e.g. caspase inhibitor), or checkpoint inhibitor is added continuously during the one or more steps of incuating the cells, such as during incubation with a T cell stimulatory agent(s). For example, after washing the cells during or after the one or more steps the culture media is replenished with the T cell adjuvant, such as at the same concentration. In some cases, the one or more T cell stimulatory agent(s) also is replenished with the wash. The washes can be carried out daily, every other day, every third day, every fourth day, every fifth day, every sixth day or once a week during the step, such as during the incubation with the one or more T cell stimulatory agent(s).

[0026] 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 the T cell stimulatory agent(s), the at least one T cell adjuvant and / or the one or more recombinant cytokines.

[0027] In some of any of the embodiments, the culturing is carried out until a threshold amount of cells is obtained and / or is carried out for up to 20 days.

[0028] In some of any of the embodiments, the population of T cells comprising tumor-reactive T cells comprise T cells surface positive for one or more T cell activation marker 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 population of T cells comprising tumor-reactive T cells is enriched for T cells surface positive for one or more T cell activation marker 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 population of T cells comprising tumor-reactive T cells is enriched for the tumor-reactive T cells.

[0029] In some of any of the provided embodiments, the T cells comprise primary T cells from a subject, optionally a human subject.

[0030] In some of any of the provided embodiments, the population of T cells comprising tumor-reactive T cells are present in an ex vivo co-culture comprising autologous T cells from a biological sample from a subject and antigen-presenting cells (APCs), said co-culturing carried out under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject. In some of any of the provided embodiments, the population of T cells comprising tumor-reactive T cells is generated by a process comprising the steps of: (a) identifying somatic mutations associated with one or more tumor-associated antigen by exome sequencing of healthy and tumor tissue from a subject; (b) identifying at least one epitope of the one or more tumor-associated antigens, optionally wherein the at least one epitope is a neoepitope; (c) isolating a population of autologous T cells from a biological sample from the subject; and (d) co-culturing the population of T cells with antigen presenting cells (APCs) that have been exposed to or contacted with one or more peptides comprising the at least one epitope of the one or more tumor-associated antigens under conditions to present one or more of the peptides on the surface of a major histocompatibility complex (MHC), thereby generating the population of T cells comprising tumor-reactive T cells reactive to the one or more peptides.

[0031] In some embodiments, exposing or contacting APCs with the one or more peptides comprises generating a mutation library of peptides comprising the one or more epitopes, optionally wherein the peptides are 8 to 32 amino acids in length, 8 to 24 amino acids in length, 8 to 18 amino acids in length, 8 to 10 amino acids in length, 10 to 32 amino acids in length, 10 to 24 amino acids in length, 10 to 18 amino acids in length, 18 to 32 amino acids in length, 18 to 24 amino acids in length or 24 to 32 amino acids in length, optionally at or about 9mers; and pulsing the APCs with the mutation 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 APCs with the one or more peptides comprises: generating DNA, optionally a minigene construct, encoding the one or more tumor-associated antigens or a portion thereof comprising the somatic mutation; in vitro transcribing the DNA into RNA; and introducing the in vitro transcribed RNA into the APCs 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 II.

[0032] In some of any of the embodiments, the co-culturing is carried out for 1 to 7 days. In some embodiments, the incubation with the at least one T cell adjuvant is carried out during at least a portion of the co-culturing. In some embodiments, the method includes further selecting from the co-culture T cells reactive to the one or more peptides, thereby enriching for the population of T cells comprising tumor-reactive T cells. In some embodiments, the enriching is carried out prior to the incubation with the at least one T cell adjuvant. In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out before the co-culturing, wherein the population of autologous T cells from the biological sample from the subject is incubated with the T cell stimulatory agent(s). In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out after the enriching for the population of T cell cells comprising tumor-reactive T cells. In some embodiments, the APCs are autologous to the subject or allogeneic to the subject.

[0033] In some of any of the provded embodiments, the co-culture ratio of antigen presenting cells to T Cells is between 20:1 and 1:1, between 15:1 and 1:1, between 10:1 and 1:1, between 5:1 and 1:1, between 2.5:1 and 1:1, between 1:20 and 1:1, between 1:15 and 1:1, between 1:10 and 1:1, between 1:5 and 1:1, or between 1:2.5 and 1:1. In some of any of the provided embodiments, the co-culture ratio of antigen presenting cells to T cells is or is about 1:1. In some of any of the provided embodiments, the co-culturing is for 2 hours to 24 hours. In some of any of the provided embodiments, the co-culturing is for at or about 6 hours.

[0034] In some of any of the provided embodiments, the subject is healthy or is not known to exhibit a disease or condition.

[0035] In some of any of the provided embodiments, the culturing comprises: isolating a population of cells comprising T cells from a biological sample from a subject; incubating the population of cells comprising T cells with a T cell stimulatory agent(s) under conditions to activate T cells of the population to produce a population of T cells comprising activated cells; co-culturing the population of T cells comprising activated cells in the presence of APCs under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population of T cells comprising tumor reactive T cells, wherein the co-culturing is carried out in the presence of the at least one T cell adjuvant thereby incubating the population of T cells comprising tumor-reactive T cells with the at least one adjuvant; selecting from the co-culture T cells reactive to the one or more peptides, thereby enriching for the population of T cells comprising tumor-reactive T cells; and further incubating the cells in the presence of one or more recombinant cytokines, optionally wherein the incubating in the presence of one or more cytokines is carried out prior to, during and / or subsequent to the incubating with the T cell stimulatory agents(s), co-culturing in the presence of APCs and / or incubating in the presence of one or more T cell adjuvants , wherein the culturing stimulates expansion of cells of the population of T cells.

[0036] In some of any of the provided embodiments, the culturing comprises:isolating a population of cells comprising T cells from a biological sample from a subject; incubating the population of cells comprising T cells with a T cell stimulatory agent(s) under conditions to activate T cells of the population to produce a population of T cells comprising activated cells; co-culturing the population of T cells comprising activated cells in the presence of APCs under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population of T cells comprising tumor reactive T cells; selecting from the co-culture T cells reactive to the one or more peptides, thereby enriching for the population of T cells comprising tumor-reactive T cells; incubating the enriched population of T cells comprising tumor-reactive cells in the presence of the 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 one or more cytokines is carried out prior to, during and / or subsequent to the incubating with the T cell stimulatory agents(s), co-culturing in the presence of APCs and / or incubating in the presence of one or more T cell adjuvants, wherein the culturing stimulates expansion of cells of the population of T cells.

[0037] In some of any of the provided embodiments, the culturing comprises:isolating a population of cells comprising T cells from a biological sample from a subject; co-culturing the population of T cells in the presence of APCs under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population of T cells comprising tumor reactive T cells; incubating the population comprising tumor-reactive T cells with a T cell stimulatory agent(s) under conditions to activate T cells of the population to produce a population of T cells comprising activated cells; selecting from the co-culture T cells reactive to the one or more peptides, thereby enriching for the population of T cells comprising tumor-reactive T cells, wherein the selecting is carried out prior to or after the incubating with the T cells stimulatory agent(s); incubating the population of T cells enriched for tumor-reactive T cellscells in the presence of the 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 one or more cytokines is carried out prior to, during and / or subsequent to the incubating with the T cell stimulatory agents(s), co-culturing in the presence of APCs and / or incubating in the presence of one or more T cell adjuvants, wherein the culturing stimulates expansion of cells of the population of T cells.

[0038] In some of any of the provided embodiments, the co-culturing is carried out for 1-7 days.

[0039] In some of any of the provided embodiments, the population of T cells comprising tumor-reactive T cells are isolated or selected directly from a biological sample from a subject. In some of any of the provided embodiments, the subject exhibits a disease or condition, optionally wherein the disease or condition is a cancer. In some of any of the provided embodiments, the subject has been previously administered a costimulatory agonist prior to the isolating or selecting of the population of T cells.

[0040] In some of any of the provided embodiments, the population of T cells comprising tumor-reactive T cells are allogeneic to a subject to be treated or autologous to a subject to be treated, optionally wherein the subject to be 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.

[0041] In some of any of the provided embodiments, the biological sample is a peripheral blood sample and the peripheral blood sample is collected by a blood draw or by apheresis, optionally wherein the apheresis is leukapheresis. In some of any of the provided embodiments, the biological sample comprises a volume of between 50 mL and 400 mL. In some of any of the provided embodiments, the enriching for the tumor reactive T cells or T cells surface positive for the one or more activation markers is by flow cytometry, optionally carried out by automated high-throughput flow cytometry In some embodiments, the enriching can be carried out using an FX500 cell sorter. In some of any of the provided embodiments, 1 run, 2 runs, 3 runs or 4 runs by flow cytometry is carried out to enrich the tumor-reactive T cells from the sample.

[0042] In some of any of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, wherein the sample is collected by a needle biopsy, optionally a core needle biopsy or a fine-needle aspiration. In some of any of the emobdiments, the biological sample includes tumor fragments. In some of any of the provided embodiments, the population of T cells comprise tumor infiltrating lymphocytes.

[0043] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) enriching tumor-reactive T cells from a biological sample comprising T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and (b) incubating the population of T cells enriched for 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 is carried out prior to, concurrently with, or subsequent to incubating the population of T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of cells of the population of T cells, wherein the culturing stimulates expansion of cells of the population of T cells; and (2) harvesting cells produced by the method.

[0044] In some embodiments, provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) enriching tumor-reactive T cells from a biological sample comprising T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (b) incubating the population of T cells enriched for tumor-reactive T cells with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; and (c) incubating the population of T cells enriched for tumor-reactive T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells, wherein at least a portion of the incubating is carried out prior to, concurrently with, or subsequent to the incubating in (b), wherein the culturing stimulates expansion of cells of the population of T cells; and (2) harvesting cells produced by the method.

[0045] In some of any of the provided embodiments, the culturing the T cells further comprises incubating the cells in the presence of one or more recombinant cytokines, wherein the incubating in the presence of one or more cytokines is carried out prior to, during and / or subsequent to the incubating with the T cell stimulatory agents(s) and / or incubating in the presence of the one or more T cell adjuvants.

[0046] In some of any of the embodiments, the T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines, wherein the incubating in the presence of the T cell stimulatory agent(s) is carried out prior to, during and / or subsequent to the incubating in the presence of the one or more T cell adjuvants.

[0047] In some of any of the embodiments, the T cell stimulating agent(s) comprises an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling via a costimulatory receptor, optionally wherein the costimulatory receptor is CD28, wherein the incubating in the presence of the T cell stimulating agent(s) is carried out prior to, during and / or subsequent to the incubating in the presence of the one or more T cell adjuvants.

[0048] 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 stimulatory agent(s), the at least one T cell adjuvant and / or the 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 is carried out for up to 20 days.

[0049] In some of any of the provided embodiments, at least a portion of the incubating the population of T cells with the at least one T cell adjuvant is carried out concurrently with the incubating the population of T cells with the T cell stimulatory agent(s).

[0050] In some of any of the provided embodiments, the incubating the population of T cells with the at least one T cell adjuvant is carried out after the incubating the population of T cells with the T cell stimulatory agent(s).

[0051] In some of any of the provided embodiments, the culturing comprises: enriching tumor-reactive T cells from a biological sample comprising T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; incubating the population of T cells comprising reactive T cells with a T cell stimulatory agent(s) under conditions to activate T cells of the population to produce a population of T cells comprising activated cells; incubating the population of T cells comprising activated T cells in the presence of the 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 one or more cytokines is carried out prior to, during and / or subsequent to the incubating with the T cell stimulatory agents(s) and / or incubating in the presence of one or more T cell adjuvants, wherein the culturing stimulates expansion of cells of the population of T cells. In some of any of the provided embodiments, the incubating in the presence of the at least one T cell adjuvant is carried out between 1 and 5 days prior to the harvesting of the cells.

[0052] In some of any of the provided embodiments, the incubating the population of T cells with the at least one T cell adjuvant is carried out prior to the incubating of the population of T cells with the T cell stimulatory agent(s). In some of any of the provided embodiments, subsequent to the incubating the population of T cells with the at least one T cell adjuvant and prior to incubating the population of T cells with the T cell stimulatory agent(s), enriching for the tumor-reactive T cells and / or the T cells expressing one or more T cell activation marker, wherein the population of enriched T cells is incubated with the T cell stimulatory agent(s).

[0053] In some of any of the provided embodiments, the T cells are primary T cells from a subject, optionally a human subject.

[0054] In some of any of the provided embodiments, the biological sample is an ex vivo co-culture comprising autologous T cells from a subject and antigen-presenting cells (APCs), said co-culturing carried out under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject. In some of any of the provided embodiments, the APCs are autologous to the subject or allogeneic to the subject. In some of any of the provided embodiments, the subject is healthy or is not known to exhibit a disease or condition.

[0055] In some of any of the provided embodiments, the biological sample is from a subject and the selecting comprises directly isolating the T cells from the biological sample. In some of any of the provided embodiments, the subject exhibits a disease or condition, optionally wherein the disease or condition is a cancer. In some of any of the provided embodiments, the subject has been previously administered a costimulatory agonist prior to the isolating or selecting of the population of T cells. In some of any of the provided embodiments, the population of T cells are allogeneic to a subject to be treated or autologous to a subject to be treated, optionally wherein the subject to be 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.

[0056] In some of any of the provided embodiments, the biological sample is a peripheral blood sample and the peripheral blood sample is collected by a blood draw or by apheresis, optionally wherein the apheresis is leukapheresis. In some of any of the provided embodiments, the biological sample comprises a volume of between 50 mL and 400 mL. In some of any of the provided embodiments, the enriching for the tumor reactive T cells or T cells surface positive for the one or more activation markers is by flow cytometry, optionally carried out by automated high-throughput flow cytometry, optionally by the FX500 cell sorter. In some of any of the provided embodiments, 1 run, 2 runs, 3 runs or 4 runs by flow cytometry is carried out to enrich the tumor-reactive T cells from the sample.

[0057] In some of any of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, wherein the sample is collected by a needle biopsy, optionally a core needle biopsy or a fine-needle aspiration. In some embodiments, the biological sample includes tumor fragments. In some of any of the provided embodiments, the population of T cells comprise tumor infiltrating lymphocytes.

[0058] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) incubating a population of T cells from a biological sample from a subject with one or more recombinant cytokines for stimulating 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 tumor-reactive T cells from the population of T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (c) incubating the population of T cells enriched for tumor-reactive T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells; and (2) harvesting cells produced by the method.

[0059] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) incubating a population of T cells with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; (b) enriching tumor-reactive T cells from the population of T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (c) incubating the population of T cells enriched for tumor-reactive T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells; and (d) further incubating the cells in the presence of one or more recombinant cytokines, optionally wherein the incubating in the presence of one or more cytokines is carried out prior to, during and / or subsequent to the incubating with the T cell stimulatory agents(s) and / or incubating in the presence of one or more T cell adjuvants, wherein the culturing stimulates expansion of cells of the population of T cells; and (2) harvesting cells produced by the method.

[0060] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) incubating a population of cells comprising T cells from a biological sample from a subject that has a tumor with a T cell stimulatory agent(s) comprising one or more recombinant cytokinesunder conditions to stimulate expansion of T cells in the population to produce a population of stimulated T cells, 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 population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (c) enriching tumor-reactive T cells from the co-culture, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (d) incubating the population of T cells enriched for tumor-reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells; and (2) harvesting the expanded cells produced by the method.

[0061] In some of any of the provided embodiments, at least one T cell adjuvant further comprises at least one apoptosis inhibitor. In some of any of the provided emdbodiments, at least one apoptosis inhibitor and the checkpoint inhibitor is carried out simultaneously, intermittently or sequentially. In some of any of the provided embodiments, at least one T cell adjuvant further comprises at least one costimulatory agonist. In some of any of the provided embodiments, at least one costimulatory agonist and the checkpoint inhibitor is carried out simultaneously, intermittently or sequentially. In some of any of the provided embodiments, the apoptosis inhibitor inhibits caspase activation or activity.

[0062] In some of any of the provided emdbodiments, the apoptosis inhibitor inhibits one or more of caspase 2, a caspase 8, a caspase 9, a caspase 10, a caspase 3, a caspase 6 or a caspase 7. In some of any of the provided embodiments, the apoptosis inhibitor is selected from the group consisting of Emricasan (IDN-6556, PF-03491390), NAIP (neuronal apoptosis inhibitory protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome binding IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8), Wedelolactone, NS3694, NSCI and Z- fluoromethyl ketone Z-VAD-FMK or a flouromethyl ketone variant thereof.. In some of any of the provided emdbodiments, the apoptosis inhibitor is a pan-caspase inhibitor that inhibits activation or activity of two or more caspases. In some of any of the provided embodiments, the apoptosis inhibitor is Z-VAD-FMK. In some of any 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.

[0063] In some of any of the provided embodiments, the apoptosis inhibitor (e.g. caspase inhibitor) is added at a concentration of between at about 0.5 µM and at about 50 µM, between at about 0.5 µM and at about 40 µM, between at about 0.5 µM and at about 30 µM, between at about 0.5 µM and at about 20 µM, between at about 0.5 µM and at about 10 µM, between at about 0.5 µM and at about 5 µM, between at about 0.5 µM and at about 1 µM, between at about 1 µM and at about 50 µM, between at about 1 µM and at about 40 µM, between at about 1 µM and at about 30 µM, between at about 1 µM and at about 20 µM, between at about 1 µM and at about 10 µM, between at about 1 µM and at about 5 µM, each inclusive. In some embodiemtns, the apoptosis inhibitor (e.g. caspase inhibitor) is added at a concentration of at or about 2 µM. In some embodiemtns, the apoptosis inhibitor (e.g. caspase inhibitor) is added at a concentration of at or about 10 µM. In some embodiemtns, the apoptosis inhibitor (e.g. caspase inhibitor) is added at a concentration of at or about 25 µM. In some of any of the provided embodiments, the apoptosis inhibitor (e.g. caspase inhibitor) is added at a concentration of between at about 0.5 µg / mL and at or about 25 µg / mL, between at or about 0.5 µg / mL and at or about 10 µg / mL, between at or about 0.5 µg / mL and at or about 5 µg / mL, between at or about 0.5 µg / mL and at or about 1 µg / mL, between at or about 1 µg / mL and at or about 25 µg / mL, between at or about 1 µg / mL and at or about 10 µg / mL, between at or about 1 µg / m L and at or about 5 µg / mL, between at or about 5 µg / mL and at or about 25 µg / mL, between at or about 5 µg / mL and at or about 10 µg / mL, or between at or about 10 µg / mL and at or about 25 µg / mL, each inclusive.

[0064] Provided herein is a method for manufacturing tumor reactive T cells, the method comprising: (1) culturing T cells by a process comprising: (a) incubating a population of T cells from a biological sample from a subject with one or more recombinant cytokines for stimulating 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) co-culturing the population of T cells produced in (a) in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (c) enriching tumor-reactive T cells produced in (b), said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (d) incubating the population of T cells enriched for tumor-reactive T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells; and (2) harvesting cells produced by the method.

[0065] 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 stimulatory agent(s), the at least one T cell adjuvant and / or the 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 is carried out for up to 30 days. In some of any of the provided embodiments, the culturing is carried out until a threshold amount of cells is obtained and / or is carried out for up to 20 days. In some of any of the provided embodiments, the population of T cells comprises primary T cells from a subject, optionally a human subject. In some of any of the provided embodiments, the population of T cells are isolated from an ex vivo co-culture comprising autologous T cells from a biological sample from a subject and antigen-presenting cells (APCs), said co-culturing carried out under conditions in which the APCs have been induced to present one or more neotantigen peptides from a tumor from the subject. In some of any of the provided embodiments, the APCs are autologous to the subject or allogeneic to the subject. In some of any of the provided embodiments, the subject is healthy or is not known to exhibit a disease or condition. In some of any of the provided embodiments, the population of T cells are isolated or selected directly from a biological sample from a subject. In some of any of the provided embodiments, the subject exhibits a disease or condition, optionally wherein the disease or condition is a cancer. In some of any of the provided embodiments, the population of T cells are allogeneic to a subject to be treated or autologous to a subject to be treated, optionally wherein the subject to be treated has a tumor or cancer.

[0066] In some of any of the provided embodiments, the biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample.

[0067] In some of any of the provided embodiments, the biological sample is a peripheral blood sample and the peripheral blood sample is collected by a blood draw or by apheresis, optionally wherein the apheresis is leukapheresis. In some of any of the provided embodiments, the biological sample comprises a volume of between 50 mL and 400 mL. In some of any of the provided embodiments, the enriching for the tumor reactive T cells or T cells surface positive for the one or more activation markers is by flow cytometry, optionally carried out by automated high-throughput flow cytometry, optionally by the FX500 cell sorter. In some of any of the provided embodiments, 1 run, 2 runs, 3 runs or 4 runs by flow cytometry is carried out to enrich the tumor-reactive T cells from the sample.

[0068] In some of any of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, wherein the sample is collected by a needle biopsy, optionally a core needle biopsy or a fine-needle aspiration. In some embodiments, the biological sample includes tumor fragments. In some of any of the provided embodiments, the population of T cells comprise tumor infiltrating lymphocytes.

[0069] In some of any of the provided embodiments, the enriching for tumor-reactive T cells comprises selecting for T cells surface positive for one or more T cell activation marker 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.

[0070] In some of any of the provided embodiments, the enriching for the tumor reactive T cells comprises selection of T cells surface positive for the one or more T cell activation markers. In some of any of the provided embodiments, the one or more T cell activation marker is 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 any of the provided embodiments, the one or more T cell activation marker is selected from the group consisting of CD38, CD39, CD6, CD90, CD134 and CD137. In some of any of the provided embodiments, the one or more T cell activation marker is CD134 and / or CD137. In some of any of the provided embodiments, the one or more T cell activation marker is 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 marker is selected from the group consisting of CD107a, CD39, CD103, CD59, CD90 and CD38.

[0071] In some of any of the provided embodiments, the one or more T cell activation marker comprises 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 marker further comprises CD137. In some of any of the provided embodiments, the one or more T cell activation marker comprises 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 T cell activation marker further comprises at least one marker selected from the group consisting of PD-1, TIM-3 and LAG-3.

[0072] In some of any of the provided embodiments, the T cells comprise CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells. In some of any of the provided embodiments, the population of T cells is enriched for CD4+ T cells and CD8+ T cells. In some of any of the provided embodiments, enriching for CD4+ and CD8+ T cells comprises selecting for T cells surface positive for the cell surface marker CD3 or comprises sequential or simultaneous selection for T cells surface positive for the cell surface marker CD4 and T cells surface positive for the cell surface marker CD8. In some of any of the provided embodiments, the population enriched for CD4+ T cell and CD8+ T cells comprises CD3+ T cells as a percentage of total cells in the population that is greater than or greater than about 60%, greater than or greater than about 70%, greater than or greater than about 80%, greater than or greater than about 90% or greater than or greater than about 95%; or the population enriched for CD4+ T cell and CD8+ T cells comprises CD4+ T cells and CD8+ T cells as a percentage of total cells in the population that is greater than or greater than about 60%, greater than or greater than about 70%, greater than or greater than about 80%, greater than or greater than about 90% or greater than or greater than about 95%. In some of any of the provided embodiments, the ratio of CD8+ T cells to CD4+ T cells is between at or about 1:100 and at or about 100:1, between at or about 1:50 and at or about 50:1, between at or about 1:25 and at or about 25: 1, between at or about 1:10 and at or about 10:1, between at or about 1:5 and at or about 5:1, or between at or about 1:2.5 and at or about 2.5:1.

[0073] In some of any of the provided embodiments, the tumor-associated antigen is a neoantigen; and / or the tumor-reactive T cells comprise T cells comprising an endogenous TCR that is reactive to a neoantigen, In some of any of the provided embodiments, the population enriched for tumor-reactive T cells comprises tumor-reactive T cells as a percentage of total cells or total T cells in the population that is greater than or greater than about 60%, greater than or greater than about 70%, greater than or greater than about 80%, greater than or greater than about 90% or greater than or greater than about 95%; and / or the population enriched for tumor-reactive T cells comprises T cells with an activation phenotype as a percentage of total cells or total T cells in the population that is greater than or greater than about 60%, greater than or greater than about 70%, greater than or greater than about 80%, greater than or greater than about 90% or greater than or greater than about 95%, wherein the activation phenotype comprises T cells surface positive for one or more T cell activation marker 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.

[0074] In some of any of the provided embodiments, the one or more T cell activation marker is 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 marker is 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 comprising selecting, from a biological sample, T cells that are surface positive for one or more T cell activation marker selected from the group consisting of CD107, CD107a, CD39, CD103, CD59, CD90, CD38, CD30, CD154, CD252, CD134, CD258 and CD256.

[0075] In some of any of the provided embodiments, further comprising incubating selected cells in the presence of at least one T cell adjuvant and / or a T cell stimulatory agent(s) under conditions for expansion or proliferation of the cells.

[0076] In some of any of the provided embodiments, the T cells are primary T cells from a subject, optionally a human subject. In some of any of the provided embodiments, the biological sample is an ex vivo co-culture comprising autologous T cells from a subject and antigen-presenting cells (APCs), said co-culturing carried out under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject. In some of any of the provided embodiments, the APCs are autologous to the subject or allogeneic to the subject. In some of any of the provided embodiments, the subject is healthy or is not known to exhibit a disease or condition.

[0077] In some of any 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 any of the provided embodiments, the antigen presenting cells are dendritic cells. In some of any 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 comprises at least one neoepitope from tumor-associated antigens from the subject.

[0078] In some of any of the provided embodiments, the biological sample is from a subject and the selecting comprises directly isolating the T cells from the biological sample. In some of any of the provided embodiments, the subject exhibits a disease or condition, optionally wherein the disease or condition is a cancer. In some of any of the provided embodiments, the subject has been previously administered a costimulatory agonist prior to the isolating or selecting of the population of T cells. In some of any of the provided embodiments, the T cells are allogeneic to a subject to be treated or autologous to a subject to be treated, optionally wherein the subject to be treated has a tumor or cancer.

[0079] In some of any of the provided embodiments, the biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample. In some of any of the provided embodiments, the biological sample is a peripheral blood sample and the peripheral blood sample is collected by a blood draw or by apheresis, optionally wherein the apheresis is leukapheresis. In some of any of the provided embodiments, the biological sample is a lymph node sample or a tumor sample, wherein the sample is collected by a needle biopsy, optionally a core needle biopsy or a fine-needle aspiration.

[0080] In some of any of the provided embodiments, the one or more T cell activation marker is 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 marker comprises 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.

[0081] In some of any of the provided embodiments, the one or more T cell activation marker further comprises CD137. In some of any of the provided embodiments, the one or more reactive T cell activation marker comprises 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 marker further comprises at least one marker selected from the group consisting of PD-1, TIM-3 and LAG-3.

[0082] In some of any of the provided embodiments, the selecting comprises immunoaffinity-based selection. In some of any of the provided embodiments, the immununoaffinity-based selection is carried out by contacting cells with an antibody that specifically binds to the marker and recovering cells bound to the antibody. In some of any of the provided embodiments, the antibody is immobilized on a particle, optionally wherein the particle is a bead or a nanoparticle, optionally wherein the particle is a magnetic particle. In some of any of the provided embodiments, the selecting comprises magnetic-based selection. In some of any of the provided embodiments, the antibody is labeled with a detectable agent, optionally wherein the detectable agent is a fluorophore. In some of any of the provided embodiments, the selecting comprises flow cytometry.

[0083] In some of any of the provided embodiments, the at least one T cell adjuvant is soluble and / or is not bound or attached to a solid support, optionally wherein the solid support is a bead. 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 apoptotic blocker.

[0084] In some of any of the provided embodiments, the incubating with the at least one costimulatory agonist and the at least one apoptotic blocker is carried out simultaneously, intermittenttly or sequentially.

[0085] In some of any of the provided embodiments, the at least one T cell adjuvant comprises at least one costimulatory agonist. In some of any of the provided embodiments, the costimulatory agonist does not specifically bind CD28 or is not an anti-CD28 antibody. In some of any of the provided embodiments, the costimulatory agonist is a tumor necrosis factor receptor superfamily (TNFRSF) agonist. In some of any of the provided embodiments, the costimulatory agonist is an antibody or antigen-binding fragment that specifically binds a TNFRSF member or is a fusion protein comprising an extracellular domain or binding portion thereof of a ligand of a TNFRSF member. In some of any of the provided embodiments, the TNFRSF member is selected from OX40, 4-1BB, GITR and CD27.

[0086] In some of any of the provided embodiments, the costimulatory agonist specifically binds OX40. In some of any 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 any of the provided embodiments, the costimulatory agonist is an OX40L fusion protein that is MEDI6383. In some of any of the provided embodiments, the costimulatory agonist is Tavolixizumab.

[0087] In some of any of the provided embodiments, the costimulatory agonist specifically binds 4-1BB. In some of any of the provided embodiments, the costimulatory agonist is urelumab or Utomilumab, or is an antigen-binding fragment of any of the foregoing.

[0088] In some of any of the provided embodiments, the costimulatory agonist specifically bind CD27.

[0089] In some of any of the provided embodiments, the costimulatory agonist is Varlilumab, or is an antigen-binding fragment of any of the foregoing. In some of any of the provided embodiments, the costimulatory agonist specifically bind GITR. In some of any of the provided embodiments, the costimulatory agonist is MK-1248, or is an antigen-binding fragment of any of the foregoing.

[0090] In some of any of the provided embodiments, the at least one T cell adjuvant comprises at least one apoptosis inhibitor. In some of any of the provided embodiments, the apoptosis inhibitor reduces apoptosis induced by CD95 (Fas), optionally wherein the apoptosis inhibitor specifically binds CD95 (Fas) or CD95 ligand (Fas ligand). In some of any of the provided embodiments, the apoptosis inhibitor is an antibody or antigen-binding fragment, optionally wherein the apoptosis inhibitor is an anti-Fas antibody or an anti-Fas ligand antibody. In some of any 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 CD95 ligand (Fas ligand) fused to an Fc immunoglobulin domain, optionally wherein the apoptosis inhibitor is APG101 or CAN008.

[0091] In some of any of the provided embodiments, the apoptosis inhibitor inhibits caspase activation or activity, optionally wherein the caspase is a caspase 2, a caspase 8, a caspase 9, a caspase 10, a caspase 3, a caspase 6 or a caspase 7, optionally wherein the caspase is a caspase 3. In some of any of the provided embodiments, the apoptosis inhibitor is selected from the group consisting of NAIP (neuronal apoptosis inhibitory protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome binding IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8). In some of any of the provided embodiments, the apoptosis inhibitor is emericasan.

[0092] In some of any of the provided embodiments, the apoptosis inhibitor is added at a concentration of between at about at or about at or about 0.5 µg / mL and at or about 25 µg / mL, between at or about 0.5 µg / mL and at or about 10 µg / mL, between at or about 0.5 µg / mL and at or about 5 µg / mL, between at or about 0.5 µg / mL and at or about 1 µg / mL, between at or about 1 µg / mL and at or about 25 µg / mL, between at or about 1 µg / mL and at or about 10 µg / mL, between at or about 1 µg / mL and at or about 5 µg / mL, between at or about 5 µg / mL and at or about 25 µg / mL, between at or about 5 µg / mL and at or about 10 µg / mL, and between at or about 10 µg / mL and at or about 25 µg / mL, each inclusive.

[0093] In some of any of the proided 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 any of the provided embodiments, the immune checkpoint is selected from PD-1 / PD-L1. In some of any of the provided embodiments, the checkpoint inhibitor is an anti-PD-1 antibody, optionally wherein the antibody is selected from Pembrolizumab, cemiplimab, nivolumab, or is an antigen-binding fragment of any of the foregoing. In some of any of the provided embodiments, the checkpoint inhibitor is Pembrolizumab. In some of any of the provided embodiments, the checkpoint inhibitor is an anti-PDL1 antibody, optionally wherein the antibody is selected from avelumab, durvalumab and atezolizumab, or is an antigen-binding fragment of any of the foregoing.

[0094] In some of any of the provided embodiments, the immune checkpoint is OX40. In some of any of the provided embodiments, the checkpoint inhibitor is an anti-OX40L antibody, optionally wherein the antibody is Oxelumab or is an antigen-binding fragment thereof.

[0095] In some of any of the provided embdiments, the immune checkpoint is CTLA-4. In some of any of the provided embodiments, the checkpoint inhibitor is an anti-CTLA-4 antibody, optionally wherein the antibody is Ipilimumab or is an antigen-binding fragment thereof.

[0096] In some of any of the provided embodiments, the checkpoint inhibitor is added at a concentration of between at about at or about at or about 0.5 µg / mL and at or about 25 µg / mL, between at or about 0.5 µg / mL and at or about 10 µg / mL, between at or about 0.5 µg / mL and at or about 5 µg / mL, between at or about 0.5 µg / mL and at or about 1 µg / mL, between at or about 1 µg / mL and at or about 25 µg / mL, between at or about 1 µg / mL and at or about 10 µg / mL, between at or about 1 µg / mL and at or about 5 µg / mL, between at or about 5 µg / mL and at or about 25 µg / mL, between at or about 5 µg / mL and at or about 10 µg / mL, and between at or about 10 µg / mL and at or about 25 µg / mL, each inclusive.

[0097] In some of any of the provided embodiments, the T cell adjuvant is added continuously during the one or more steps of the culturing, wherein the T cell adjuvant is replenished or replaced one or more times during the culturing. In some of any of the provided embodiments, the T cell adjuvant is added continuously during the incubation with the one or more recombinant cytokines, wherein 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 the one or more steps of the culturing, wherein the T cell adjuvant is added only one time during the one or more steps of culturing.

[0098] In some of any of the provided embodiments, the T cell stimulatory agent(s) is selected from an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling via a costimulatory receptor, optionally wherein the costimulatory receptor is CD28. In some of any of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, optionally OKT3. In some of any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor comprises peripheral blood mononuclear cells (PBMCs), optionally non-dividing or irradiated PBMCs. In some of any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor is an anti-CD28 antibody. In some of any of the provided embodiments, the T cell stimulatory agent(s) is an anti-CD3 antibody and an anti-CD28 antibody that each are soluble.

[0099] In some embodiments, the 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, optionally wherein the recombinant cytokine is selected from one or more of IL-2, IL-15, IL-7 and IL-21.

[0100] In some of any of the provided embodiments, the second T cell stimulatory agent(s) comprise 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.

[0101] In some of any of the provided embodiments, the T cell stimulatory agent(s) is or includes recombinant IL-2.

[0102] In some of any of the provded embodiments, the one or more recombinant cytokine comprises recombinant IL-2.

[0103] Provided herein is a method for manufacturing tumor-reactive T cells, the method comprising (1) culturing T cells by a process that comprises: (a) incubating a population of cells comprising T cells from a biological sample obtained from a subject that has a tumor with a first T cell stimulatory agent(s) comprising one or more recombinant cytokine selected from one or more of IL-7, IL-15 and IL-21 under conditions to stimulate expansion of T cells of the population to produce a population of stimulated T cells; (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (d) enriching from the co-culture the population of tumor reactive T cells reactive to the one or more peptides, wherein said tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and (d) incubating the population of T cells enriched in tumor reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of T cells in the population; and (2) harvesting the expanded cells produced by the method.

[0104] In some of any of the provided embodiments, the first T cell stimulatory agent(s) comprises one or more recombinant cytokines comprises IL-7 and IL-15. In some of any of the provided embodiments, the first T cell stimulatory agent(s) further comprises recombinant IL-2. In some of any of the provided embodiments, the first T cell stimulatory agent(s) does not comprise recombinant IL-2.

[0105] In some of any of the provided embodiments, the second T cell stimulatory agent(s) comprise one or more 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. 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 cytokine comprises recombinant IL-2.

[0106] In some of any of the provided embodiments, the concentration of each of the one or more recombinant cytokine individually is 100 IU / mL to 6000 IU / mL. In some of any of the provided embodiments, the concentration of each of the one or more recombinant cytokine individually is from 300 IU / mL to 1000 IU / mL, optionally wherein the concentration of each of the one or more recombinant cyokine is at or about 300 IU / mL or is at or about 1000 IU / mL.

[0107] In some of any of the provided embodiments, the second T cell stimulatory agent(s) further comprises an anti-CD3 antibody, optionally OKT3, optionally wherein the concentration of the anti-CD3 antibody is at or about 50 ng / mL.

[0108] 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 antigen by exome sequencing of healthy and tumor tissue from a subject; (b) identifying at least one neoepitope of the 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 population of T cells with antigen presenting cells (APCs) that have been exposed to or contacted with the one or more peptides, said one or more peptides comprising the at least one neoepitope of the one or more tumor-associated antigens under conditions to present one or more of the peptides on the surface of a major histocompatibility complex (MHC), thereby generating the population of T cells comprising tumor-reactive T cells reactive to the one or more peptides.

[0109] In some of any of the provided embodiments, the MHC molecule is a class I molecule. In some of any of the provided embodiments, the MHC molecule is a Class II molecule. In some of any of the provided embodiments, the MHC molecule is MHC class I and II.

[0110] In some of any of the provided embodiments, the T cells are CD4+ cells. In some of any of the provided embodiments, the T cells are CD8+ cells. In some of any of the provided embodiments, the T cells are CD4+ cells and CD8+ cells.

[0111] In some of any of the provided embodiments, the one or more peptide comprises an individual peptide or a pool of peptides. In some of any of the provided embodiments, the one or more peptides are loaded on antigen presenting cells by transfection of in vitro transcribed synthesized minigene constructs encoding for the one or more peptides, optionally wherein the one or more peptides are flanked on each side by 12 amino acids from endogenous proteins, in tandem, wherein the transcribed minigene constructs generate individual peptides. In some of any of the provided embodiments, the one or more peptides are loaded on antigen presenting cells by peptide pulse, optionally by electroporation. In some of any of the provided embodiments, the one or more peptide is each individually 5-30 amino acids, optionally 12-25 amino acids, optionally at or about 25 amino acids in length.

[0112] In some of any the provided embodiments, the one or more peptides are a pool of peptides and the concentration of peptides in the pool of peptides for the peptide pulse is between at or about 0.001 µg / mL and at or about 40 µg / mL, 0.01 µg / mL and at or about 40 µg / mL, at or about 0.1 µg / mL and at or about 40 µg / mL, at or about 1 µg / mL and at or about 40 µg / mL, at or about 0.01 µg / mL and at or about 10 µg / mL or at or about 1 µg / mL and at or about 10 µg / mL; or the one or more peptides is an individual peptide and the concentration of individual peptides for the peptide pulse is between at or about 0.00001 µg / mL and at or about 1 µg / mL, at or about 0.00001 µg / mL and at or about 0.1 µg / mL, at or about 0.00001 µg / mL and at or about 0.01 µg / mL, at or about 0.0001 µg / mL and at or about 1 µg / mL, at or about 0.0001 µg / mL and at or about 0.1 µg / mL, at or about 0.0001 µg / mL and at or about 0.1 µg / mL or at or about 0.0001 µg / mL and at or about 0.01 µg / mL. In some of any of the provided embodiments, the concentration of individual peptides of the one or more peptide, on average, is from at or about 0.00001 µg / mL to at or about 0.01 µg / mL. In some of any of the provided embodiments, the concentration of individual peptide of the one or more peptide, on average, is from at or about 0.0001 µg / mL and at or about 0.001 µg / mL.

[0113] In some of any of the provided embodiments, harvesting is carried out within 20 days after initiation of the culturing and / or the enriching of T cells comprising tumor-reactive cells. In some of any of the provided embodiments, the culturing is carried out in the presence of a recombinant cytokine selected from the group consisting of IL-2, IL-15, IL-7 and IL-21.

[0114] 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 the initiation of the culturing.

[0115] In some of any of the provided embodiments, the culturing is carried out until a threshold amount of cells is achieved that is between at or about 0.5 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between 0.5 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 8 x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 3.5x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 1 x 10 8< total cells or total viable cells, between 1 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 1 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between 1 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 1 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 1 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 1 x 10 8< and at or about 8 x 10 8< total cells or total viable cells, between at or about 1 x 10 8< and at or about 3.5x 10 8< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 8 x 10 8< total cells or total viable cells, between at or about 8 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 8 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 8 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 8 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 8 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 15 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 15 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 15 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 15 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 60 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 60 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 60 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 12 x 10 9< and at or about 50 x 10 9< total cells or total viable cells, between at or about 12 x 10 9< and at or about 30 x 10 9< total cells or total viable cells, or between at or about 30 x 10 9< and at or about 60 x 10 9< total cells or total viable cells, each inclusive.

[0116] In some of any of the provided embodiments, the method results in a fold-expansion of T cells or in a fold-expansion of tumor reactive T cells that is at least at or about 2-fold, at least at or about 5-fold, at least at or about 10-fold, at least at or about 25-fold, at least at or about 50-fold, at least at or about 100-fold, at least at or about 250-fold, at least at or about 500-fold, at least at or about 1000-fold, or more.

[0117] In some of any of the provided embodiments, the biological sample is a peripheral blood sample, optionally an apheresis sample, and wherein: the number of cells at the initiation of the culturing is between at or about 1 x 10 9< and 7 x 10 9< total viable cells; or is at or about 1 x 10 9< total viable cells, at or about 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< 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 culturing is between at or about 0.02% and at or about 40%, at or about 0.02% and at or about 24%, at or about 0.02% and at or about 18%, at or about 0.02% and at or about 0.9% or at or about 0.02% and at or about 6.0%; and / or the number of T cells surface positive for the T cell activation marker at the initiation of the culturing is between at or about 0.1 x 10 6< and at or about 60 x 10 6< T cells, 0.1 x 10 6< and at or about 8 x 10 6< T cells, 0.1 x 10 6< and at or about 20 x 10 6< T cells, 0.3 x 10 6< and at or about 35 x 10 6< T cells or 0.3 x 10 6< and at or about 60 x 10 6< T cells; or is at or about 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 foregoing.

[0118] In some of any of the provided embodiments, the biological sample is a lymph sourced sample or a tumor sourced sample, and wherein: the number of cells at the initiation of the culturing is between at or about 10 x 10 6< and 100 x 10 6< total viable cells, 20 x 10 6< and 100 x 10 6< total viable cells, or 12 x 10 6< and 43 x 10 6< total viable cells; or is at or about 10 x 10 6< total viable cells, at or about 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 culturing is between at or about 1% and at or about 90%, at or about 1% and at or about 75%, at or about 1% and at or about 50%, at or about 1% and at or about 25% or at or about 1% and at or about 14%; and / or the number of T cells surface positive for the T cell activation marker at the initiation of the culturing is between at or about 0.7 x 10 6< and at or about 15 x 10 6< T cells, 1 x 10 6< and at or about 15 x 10 6< T cells, or at or about 0.7 x 10 6< and at or about 5.4 x 10 6< T cells; or is at or about 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 foregoing.

[0119] In some of any of the provided embodiments, the population of cells comprising T cells comprise tumor infiltrating lymphocytes, lymph lymphocytes or peripheral blood mononuclear cells.

[0120] In some of any of the provided embodiments, the biological sample is a tumor and the population of cells comprising T cells comprise tumor infiltrating lymphocytes. In some of any of the provided embodiments, the biological sample is a resected tumor and the population of cells comprising T cells are one or more tumor fragments from the resected tumor. In some of any of the provided embodiments, the one or more tumor fragments are seeded for incubation with the first T cell stimulatory agent(s) at about 1 tumor fragment per 2 cm 2< . In some of any of the provided embodiments, the tumor is a melanoma. In some of any of the provided embodiments, the biological sample is a resected tumor and the population of cells comprising T cells are 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 any of the provided embodiments, the biological sample is a resected tumor and the population of cells comprising T cells are a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from the resected tumor. In some of any of the provided embodiments, the enzymatic digestion is by incubation with a collagenase, optionally collagenase IV or collagenase I / II.

[0121] In some of any of the provided embodiments, the population of cells comprising T cells are seeded for incubation with the first T cell stimulatory agent(s) at about 5 x 10 5< to at or about 2 x 10 6< total cells per 2 cm 2< . In some of any of the provided embodiments, the tumor is a colorectal cancer (CRC).

[0122] In some of any of the provided embodiments, the incubation with the at least one T cell adjuvant is for a minimum of 24 hours and up to the time of harvest of the cells, or is between at or about 24 hours and at or about 96 hours or is between at or about 24 hour and at or about 48 hours, or is at or about 24 hours, at or about 36 hours or at or about 48 hours.

[0123] In some of any of the provided embodiments, the incubation with the T cell stimulatory agent(s) is between at or about 24 hours and at or about 96 hours or is between at or about 24 hour and at or about 48 hours, or is at or about 24 hours, at or about 36 hours or at or about 48 hours.

[0124] In some of any of the provided embodiments, the incubating with the first T cell stimulatory agent(s) is for 7 to 21 days, optionally 7 to 14 days. In some of any of the provided embodiments, the incubating with the first T cell stimulatory agent(s) is in a closed system. In some of any of the provided embodiments, the incubating with the first T cell stimulatory agent(s) is in a gas permeable culture vessel. In some of any of the provided embodiments, the incubating with the first T cell stimulatory agent(s) is performed using a bioreactor. In some of any of the provided embodiments, the incubating with the second T cell stimulatory agent(s) is for 7 to 21 days, optionally 7 to 14 days. In some of any of the provided embodiments, the incubating with the second T cell stimulatory agent(s) is in a closed system. In some of any of the provided embodiments, the incubating with the second T cell stimulatory agent(s) is in a gas permeable culture vessel. In some of any of the provided embodiments, the incubating with the second T cell stimulatory agent(s) is performed using a bioreactor.

[0125] In some of any of the provided embodiments, one or more of the steps of the method is carried out in a closed system, optionally wherein one or more of the steps for selection and / or enrichment or incubation is carried out in a closed system.

[0126] In some of any of the provided embodiments, the method further comprises formulating the harvested cells for administration to a subject. In some of any of the provided embodiments, the formulating comprises cryopreservation, wherein the cells are thawed prior to administration to the subject.

[0127] Provided herein is a composition produced by any of the provided methods.

[0128] Provided herein is a composition comprising tumor-reactive T cells, wherein at least at or about 40%, at least at or about 50%, at least at or about 60%, at least at or about 70%, at least at or about 80%, or at least at or about 90% of the total cells or total T cells in the composition are tumor reactive T cells or are surface positive for one or more T cell activation marker.

[0129] Provided herein is a composition comprising expanded T cells enriched in tumor reactive T cells produced by any of the provided methods.

[0130] Provided herein is a composition comprising a pharmaceutically acceptable excipient produced by any of the provided methods.

[0131] In some of any of the embodiments of the provided compositions, the one or more T cell activation marker is 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 marker is 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 marker is selected from the group consisting of CD107a, CD39, CD103, CD59, CD90 and CD38. In some embodiments, the one or more T cell activation marker comprises 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, one or more T cell activation marker further comprises CD137. In some embodiments, the one or more reactive T cell activation marker comprises 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 embodiments. the one or more reactive T cell marker further comprises at least one marker selected from the group consisting of PD-1, TIM-3 and LAG-3.

[0132] In some of any of the embodiments 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, wherein the ratio of CD8+ T cells to CD4+ T cells is between at or about 1: 100 and at or about 100: 1, between at or about 1:50 and at or about 50:1, between at or about 1:25 and at or about 25: 1, between at or about 1:10 and at or about 10: 1, between at or about 1:5 and at or about 5:1, or between at or about 1:2.5 and at or about 2.5:1.

[0133] In some of any of the provided embodiments, the method results in a fold-expansion of T cells or in a fold-expansion of tumor reactive T cells that is at least at or about 2-fold, at least at or about 5-fold, at least at or about 10-fold, at least at or about 25-fold, at least at or about 50-fold, at least at or about 100-fold, at least at or about 250-fold, at least at or about 500-fold, at least at or about 1000-fold, or more.

[0134] In some of any of the provided embodiments, the composition of expanded cells are able to produce IFNgamma at a concentration of greater than at or about 30 pg / mL, optionally greater than at or about 60 pg / mL, following antigen-specific stimulation. In some of any of the provided embodiments, the method comprises formulating the harvested cells with a cryoprotectant. ,

[0135] In some of any of the provided embodiments, the T cells are CD3+ T cells or comprise CD4+ T cells and / or CD8+ T cells. In some of any of the provided embodiments, the T cells comprise CD4+ T cells and CD8+ T cells, wherein the ratio of CD8+ T cells to CD4+ T cells is between at or about 1:100 and at or about 100:1, between at or about 1:50 and at or about 50:1, between at or about 1:25 and at or about 25:1, between at or about 1:10 and at or about 10:1, between at or about 1:5 and at or about 5:1, or between at or about 1:2.5 and at or about 2.5:1.

[0136] In some of any of the provided embodiments, the number of tumor reactive T cells or total T cells surface positive for the T cell activation marker, or of viable cells thereof, in the composition is between at or about 0.5 x 10 8< and at or about 50 x 10 9< , between at or about 0.5 x 10 8< and at or about 30 x 10 9< , between 0.5 x 10 8< and at or about 12 x 10 9< , between at or about 0.5 x 10 8< and at or about 60 x 10 8< , between at or about 0.5 x 10 8< and at or about 15 x 10 8< , between at or about 0.5 x 10 8< and at or about 8 x 10 8< , between at or about 0.5 x 10 8< and at or about 3.5x 10 8< , between at or about 0.5 x 10 8< and at or about 1 x 10 8< , between 1 x 10 8< and at or about 50 x 10 9< , between at or about 1 x 10 8< and at or about 30 x 10 9< , between 1 x 10 8< and at or about 12 x 10 9< , between at or about 1 x 10 8< and at or about 60 x 10 8< , between at or about 1 x 10 8< and at or about 15 x 10 8< , between at or about 1 x 10 8< and at or about 8 x 10 8< , between at or about 1 x 10 8< and at or about 3.5x 10 8< , between at or about 3.5 x 10 8< and at or about 50 x 10 9< , between at or about 3.5 x 10 8< and at or about 30 x 10 9< , between at or about 3.5 x 10 8< and at or about 12 x 10 9< , between at or about 3.5 x 10 8< and at or about 60 x 10 8< , between at or about 3.5 x 10 8< and at or about 15 x 10 8< , between at or about 3.5 x 10 8< and at or about 8 x 10 8< , between at or about 8 x 10 8< and at or about 50 x 10 9< , between at or about 8 x 10 8< and at or about 30 x 10 9< , between at or about 8 x 10 8< and at or about 12 x 10 9< , between at or about 8 x 10 8< and at or about 60 x 10 8< , between at or about 8 x 10 8< and at or about 15 x 10 8< , between at or about 15 x 10 8< and at or about 50 x 10 9< , between at or about 15 x 10 8< and at or about 30 x 10 9< , between at or about 15 x 10 8< and at or about 12 x 10 9< , between at or about 15 x 10 8< and at or about 60 x 10 8< , between at or about 60 x 10 8< and at or about 50 x 10 9< , between at or about 60 x 10 8< and at or about 30 x 10 9< , between at or about 60 x 10 8< and at or about 12 x 10 9< , between at or about 12 x 10 9< and at or about 50 x 10 9< , between at or about 12 x 10 9< and at or about 30 x 10 9< , or between at or about 30 x 10 9< and at or about 60 x 10 9< , each inclusive.

[0137] In some of any of the embodiments of the provided compositions, the number of tumor reactive T cells or total T cells surface positive for the T cell activation marker, or of viable cells thereof, in the composition is between at or about 0.5 x 10 8< and at or about 50 x 10 9< , between at or about 0.5 x 10 8< and at or about 30 x 10 9< , between 0.5 x 10 8< and at or about 12 x 10 9< , between at or about 0.5 x 10 8< and at or about 60 x 10 8< , between at or about 0.5 x 10 8< and at or about 15 x 10 8< , between at or about 0.5 x 10 8< and at or about 8 x 10 8< , between at or about 0.5 x 10 8< and at or about 3.5x 10 8< , between at or about 0.5 x 10 8< and at or about 1 x 10 8< , between 1 x 10 8< and at or about 50 x 10 9< , between at or about 1 x 10 8< and at or about 30 x 10 9< , between 1 x 10 8< and at or about 12 x 10 9< , between at or about 1 x 10 8< and at or about 60 x 10 8< , between at or about 1 x 10 8< and at or about 15 x 10 8< , between at or about 1 x 10 8< and at or about 8 x 10 8< , between at or about 1 x 10 8< and at or about 3.5x 10 8< , between at or about 3.5 x 10 8< and at or about 50 x 10 9< , between at or about 3.5 x 10 8< and at or about 30 x 10 9< , between at or about 3.5 x 10 8< and at or about 12 x 10 9< , between at or about 3.5 x 10 8< and at or about 60 x 10 8< , between at or about 3.5 x 10 8< and at or about 15 x 10 8< , between at or about 3.5 x 10 8< and at or about 8 x 10 8< , between at or about 8 x 10 8< and at or about 50 x 10 9< , between at or about 8 x 10 8< and at or about 30 x 10 9< , between at or about 8 x 10 8< and at or about 12 x 10 9< , between at or about 8 x 10 8< and at or about 60 x 10 8< , between at or about 8 x 10 8< and at or about 15 x 10 8< , between at or about 15 x 10 8< and at or about 50 x 10 9< , between at or about 15 x 10 8< and at or about 30 x 10 9< , between at or about 15 x 10 8< and at or about 12 x 10 9< , between at or about 15 x 10 8< and at or about 60 x 10 8< , between at or about 60 x 10 8< and at or about 50 x 10 9< , between at or about 60 x 10 8< and at or about 30 x 10 9< , between at or about 60 x 10 8< and at or about 12 x 10 9< , between at or about 12 x 10 9< and at or about 50 x 10 9< , between at or about 12 x 10 9< and at or about 30 x 10 9< , or between at or about 30 x 10 9< and at or about 60 x 10 9< , each inclusive.

[0138] In some of any of the provided embodiments, the therapeutically effective dose is between 1 x 10 9< and 10 x 10 9< T cells.

[0139] In some of any of the embodiments of the provided compositions, the composition further contains a pharmaceutically acceptable excipient. In some embodiments the composition includes a cyroprotectant.

[0140] In some of any of the embodiments of the provided compositions, the composition is is sterile.

[0141] Provided herein is a method of treatment, comprising administering to a subject having a tumor a therapeutic dose of the composition produced by any of the provided methods.

[0142] Provided herein is a method of treatment, comprising administering any of the provided compositions to a subject having a cancer. In some embodiments, the cells of the administered composition are autologous to the subject. In some embodiments, the cancer is an epithelial cancer.

[0143] In some embodiments, the cancer is melanoma, lung squamous, lung adenocarcinoma, bladder cancer, lung small cell cancer, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, stomach 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 cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer.

[0144] In some of any of the provided embodiments, a composition of comprising expanded cells produced by the method are used to treat the cancer in the subject. In some of any of the provided embodiments, the tumor is a tumor of an epithelial cancer. In some of any of the provided methods, the tumor is a tumor of a melanoma, lung squamous, lung adenocarcinoma, bladder cancer, lung small cell cancer, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, stomach cancer or uterine cancer. In some of any of the provided methods, the tumor is a tumor of a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma cancer, endometrial cancer, optionally wherein the breast cancer is HR+ / Her2- breast cancer, triple negative breast cancer (TNBC) or HER2+ breast cancer.Brief Description of the Drawings

[0145] FIG. 1A depicts a schematic of an exemplary process for manufacturing a T cell therapeutic composition in accord with 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-culturing methods with autologous T cells obtained from the same subject. In some cases, a population of T cells from the patient, e.g. containing tumor infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL), is stimulated under conditions to expand the cells prior to co-culture with antigen presenting cells that have been contacted or exposed to peptide neoepitopes for presentation on a major histocompability complex. Following co-culture under conditions in which the antigen presenting cells present peptides in the context of a major histocompatibility complex, tumor-reactive T cells or T cells surface positive for one or more T cell activation marker (e.g. CD70a) associated with tumor reactive T cells can be selected and cultured under conditions for expansion in accord with the provided methods, such as incubation with a T cell stimulatory agent(s) (e.g. anti-CD3 / anti-CD28). The steps can include incubation with a T cell adjuvant that is a costimulatory agonist (e.g. OX40 or 4-1BB agonist) or an apoptosis inhibitor (e.g. Fas / Fas ligand inhibitor or caspase inhibitor) in accord with the provided methods.. The culturing can be carried out in the presence of one or more recombinant cytokines (e.g. IL-2) to support proliferation and expansion of cells. The process can be carried out in the presence of serum-free media containing nutrients. One or more or all of the steps can be carried out in a closed system, such as without exposure of cells to the environment. Upon reaching a therapeutic dose or a threshold number of cells, the cells can be harvested and formulated, in some cases concentrated or cryopreserved, and used for administration to a subject such as by infusion. FIG. 1B depicts a schematic of an exemplary process for manufacturing a T cell therapeutic composition in accord with the provided methods. In the exemplary process, a biological sample containing T cells is used as a cellular source for the methods. The biological sample can include tumor infiltrating lymphocytes, peripheral blood mononuclear cells (e.g. apheresis), or lymph sourced lymphocytes. Tumor-reactive T cells or T cells surface positive for one or more T cell activation marker (e.g. CD70a) associated with tumor reactive T cells can be selected directly from the sample and cultured under conditions for expansion in accord with 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. Fas / Fas ligand inhibitor or caspase inhibitor), and incubation with a T cell stimulatory agent(s) (e.g. anti-CD3 / anti-CD28). The culturing can be carried out in the presence of one or more recombinant cytokines (e.g. IL-2) to support proliferation and expansion of cells. The process can be carried out in the presence of serum-free media containing nutrients. One or more or all of the steps can be carried out in a closed system, such as without exposure of cells to the environment. Upon reaching a therapeutic dose or a threshold number of cells, the cells can be harvested and formulated, in some cases concentrated or cryopreserved, and used for administration to a subject such as by infusion. FIG. 1C depicts a full process flow chart for the generation of a population of patient specific tumor-derived infiltrating T cells. FIG. 2A depicts exemplary kinetics and T cell neoantigen reactivity in a typical TIL expansion process involving a bulk expansion of T cells with a first initial expansion and a second rapid expansion wherein reactivity remains low throughout the process, including within the final product. FIG. 2B further depicts the exemplary kinetics of a TIL expansion process involving a first initial expansion, followed by an 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 enriched reactive cells. FIG. 3A depicts the generation of total viable Population 1 cells from patient derived CRC tumor tissue using fragment culture, homogenization with enzyme, and homogenization without enzyme. Digestion with and without enzyme both yielded more total cells than culture from fragments. Percent viability of these cells is shown in FIG. 3B. Viabilities of cultures generated from fragments and digested with enzyme were higher than those derived using homogenization without enzyme. FIG. 4A depicts the generation of Population 1 cells from patient derived melanoma tumor tissue using fragment culture or homogenization with or without enzyme. Fragment culture yielded more total cells than cultures initiated from single cell suspensions. Percent viability of these cells is shown in FIG. 4B. The population generated from fragments showed higher viability than cells from single cell suspensions. FIG. 5 depicts growth curves ( FIG. 5A) as well as fold expansion ( FIG. 5B) of Population 2 cells derived from primary CRC tumors in either a conventional 6-well culture plate or a 24-well gas permeable culture plate. FIG. 5 also depicts total cell number ( FIG. 5C) as well as fold expansion ( FIG. 5D) of Population 2 cells derived from primary CRC tumors contrasted by cellular extraction method, either fragment or single cell suspension culture. FIG. 6 depicts growth curves ( FIG. 6A) as well as fold expansion ( FIG. 6B) of Population 2 cells derived from primary melanoma tumors in either a 6-well culture plate or a 24-well gas permeable culture plate. FIG. 7 depicts total cell number ( FIG. 7A) as well as fold expansion ( FIG. 7B) of Population 2 cells derived from primary CRC tumors using serum free OpTmizer or RPMI media supplemented with 5% human serum. Similarly, FIG. 8 depicts total cell number ( FIG. 8A) as well as fold expansion ( FIG. 8B) of Population 2 cells derived from primary melanoma tumors using serum free OpTmizer or RPMI media supplemented with 5% human serum. FIG. 9 depicts total cell number ( FIG. 9A) as well as fold expansion ( FIG. 9B) of Population 2 cells derived from CRC tumors and cultured in media supplemented with either a low concentration (300 IU / mL) or a high concentration (600 IU / mL) of recombinant human IL-2. These data are similarly depicted for melanoma tumor derived cells in FIG. 10A-B. A high concentration of IL-2 was not observed to be necessary for cellular expansion. FIG. 11A depicts Population 2 total cell number and FIG. 11B depicts fold expansion from melanoma derived cell cultures that were unstimulated or stimulated with OKT3, an anti-CD3 monoclonal antibody, were observed to be largely similar. FIG. 12A-Cdepict percent upregulation of activation markers on CD8+ T cells, CD38 and CD39 ( FIG. 12A), CD134 and CD137 ( FIG. 12B), and CD69 and CD90 ( FIG. 12C), between 0 and 48 hours after activation with OKT3. FIG. 13A-Cdepict percent upregulation of activation markers on CD4+ T cells, CD38 and CD39 ( FIG. 13A), CD134 and CD137 ( FIG. 13B), and CD69 and CD90 ( FIG. 13C), between 0 and 48 hours after activation with OKT3. FIG. 14 depicts expression of selected exemplary markers in a single cell suspension culture generated from a CRC tumor on Day 0. FIG. 15A-Edepict CD3+ cell purity as a percent of Population 1 cells. FIG. 15A depicts the purity of cells from Day 0 SCS from a CRC tumor after homogenization without enzyme, with 1 mg / ml (low) enzyme, and 5 mg / ml (high) enzyme. These data are similarly shown for a melanoma derived culture in FIG. 15B. FIG. 15C depicts the purity of CD3+ Population 1 cells from Day 0 (baseline SCS) and Day 6 from fragments cultured in the presence or absence of OKT3 stimulation. FIG. 15D shows the relative purity of CD3+ cells from a CRC donor on Day 11 using fragments cultured in media supplemented with either 6000 IU / mL (high) or 300 IU / mL (low) recombinant IL-2. FIG. 15E depicts Population 1 cells (Day 9) from fragments cultured in either serum free OpTmizer media or RPMI with either OKT3 stimulation and / or IL-2 at high or low concentrations. These observations support that SCSs from tumor biopsies of CRC patients may be more capable of providing a greater number of T cells for expansion than cells obtained from culture of tumor fragments. FIG. 16 depicts the purity of CD3+ Population 1 cells derived from a melanoma patient as fragment cultures from Day 9 at high and low IL-2 concentrations and with serum containing RPMI medium or serum free OpTmizer. FIG. 17A depicts the generation of Population 3 cells following co-culture with dendritic cells loaded with peptide at concentrations from 0.1 ng / mL to 20 ng / mL. FIG. 17B depicts the fold increase in the same experiment from T cells which were co-cultured with unloaded dendritic cells ( FIG. 17B). FIG. 18A compares stimulation with one peptide or two peptides reported as % 41BB / OX40 expression. FIG. 18B depicts stimulation with one peptide or two peptides reported as fold increase from T cells which were unactivated. FIG. 19A compares two T cell to dendritic cell ratios, 1:1 and 1:2, reported as % 41BB / OX40 expression. FIG. 19B compares two T cell to dendritic cell ratios, 1:1 and 1:2, reported as fold increase from T cells which were unactivated. FIG. 20A depicts percent neoantigen reactive TCR before and after co-culture with autologous neoantigen peptides and sorting of T cells sourced from the peripheral blood of three healthy donors. FIG. 20B depicts average class I reactivity pre- and post-co-culture and sorting of CD8+ cells. FIG. 21A and FIG. 21B depict recovery from cell sorting using the Sony FX500 as both total cell input and output for two independent runs ( FIG. 21A) and percent recovery ( FIG. 21B). FIG. 22 depicts purity and gating of a CD4+ population from cell sorting using Sony FX500. The results demonstrate a high recovery of cells after selection and sorting of cells positive for upregulation markers. FIG. 23A- FIG. 23C depict expansion of tumor infiltrating T lymphocytes after sorting. FIG. 23A depicts total cell number and FIG. 23B depicts fold expansion, of Population 5 cells derived from Population 4 cells following co-culture with or without dendritic cells loaded with wild-type peptide, tumor associated peptide, or no peptide. Projected cell numbers after expansion of Population 4 cells into Population 5 cells at various cell recovery numbers post-sort are shown in FIG. 23C. FIG. 24A depicts measured IFN-gamma secretion within a bulk co-culture, positive sorted (selected) population by expression of CD137 and / or CD134 from bulk co-culture cells (enriched), or negative sorted (unselected) population form bulk co-culture cells, following stimulation with mutant (mut) peptide or normal, wild-type (WT) peptide from an ovarian cancer patient. FIG. 24B depicts enrichment of neoantigen specific population of the tumor-reactive specific cells in the positive sort and negative sort compared to the bulk unsorted T cells. FIG. 24C depicts the number of TCR clonotypes present in the unselected and selected populations and demonstrates that the diversity of incoming TCRs is high in the unsorted T cell population and that there is enrichment of unique TCR clones in the selected population. FIG. 24D depicts the pre- and post-sort cell populations from Sample A which were observed to contain CD4+ and CD8+ cells, indicating that class I and class II reactive cells are present in the enriched population. FIG. 25A depicts measured IFN-gamma secretion within a bulk co-culture, positive sorted (selected) population by expression of CD137 and / or CD134 from bulk co-culture cells (enriched), or negative sorted (unselected) population from bulk co-culture cells, following stimulation with anti-CD3 (OKT3) from colorectal cancer patient. FIG. 25B depicts enrichment of neoantigen specific population of the tumor-reactive specific cells in the positive sort and negative sort compared to the bulk unsorted T cells. FIG. 25C depicts the TCR clonality profile present in the unselected and selected populations. FIG. 25D depicts the pre- and post-sort cell populations which were observed to contain CD4+ and CD8+ cells, indicating that class I and class II reactive cells are present in the enriched population. FIG. 26A depicts enrichment of neoantigen specific population of tumor-reactive specific cells in a bulk co-culture, positive sorted (selected) population by expression of CD137 and / or CD134 from bulk co-culture cells (enriched), or negative sorted (unselected) population fromm bulk co-culture cells. FIG. 26B depicts the TCR clonality profile present in the unselected and selected populations. FIG. 26C depicts Pre- (bulk) and post-sort cell populations, which were observed to contain both CD4+ class I reactive and CD8+ class II reactive cells. Total viable CD3+ cell count for cells grown in the presense of numerous T cell adjuvants are shown with (FIG. 27A-C) and without (FIG. 28A-C) supplemental OKT3 stimulation. The results shown are for the following adjuvants: Tavolixizumab, Oxelumab, Ipilimumab, Tocilizumab, Urelumab, Pembrolizumab, Varlilumab, anti-GITR MK-1248, anto-human FasL at 10µg / mL; 25 µM for Z-VAD-FMK pan-caspase inhibitor; 250 nM for HSP inhibitor NVP-HSP990; and 1000 IU / mL for cytokine (IL-7, IL-15 or IL-21). FIG. 29 shows dose response curves for IL-7 ( FIG. 29A) and IL-15 ( FIG. 29B). Total cell number and and cell viability for cells derived from each of three healthy donors and grown in experimental conditions as described are shown in FIG.30A-B- FIG.32A-B. It was observed that cells grown in the presense of continuous caspase inhibition showed superior growth despite inherent donor variability. Cellular viability for a single activation with anti-CD3 / anti-CD28 (transient activation) treatment groups for two donors are shown in FIG. 33A-B,and total cell number for the same treatments are shown in FIG. 34A-B.Cellular viability for the continuous activation with anti-CD3 / anti-CD28 treatment groups for two donors are shown in FIG 35A-B,and total cell number for the same treatments are shown in FIG. 36A-B. FIG. 37A-Cshows the fold expansion ( FIG. 37A), total viable cells ( FIG. 37B) and percent viability ( FIG. 37C), of both SCS and tumor fragment derived cultures grown in the presence or absence of pan-caspase inhibitor Z-VAD-FMK. T cell phenotype is shown for CD3+ ( FIG. 38A-D),CD4+ ( FIG. 39A-D)and CD8+ ( FIG. 40A-D)cells grown in the presense of Ipilimumab (anti-CTLA4), Pembrolizumab (anti-PD1), Tavolixizumab (anti-TNFRSF4), Urelumab (anti-CD137), and Varlilumab (anti-CD27) at varying concentrations. Detailed Description

[0146] Provided herein is a method for manufacturing T cells that express a cell surface receptor that recognizes peptides on the surface of a target cells, such as a tumor. The T cells can be tumor-reactive T cells that recognize tumor-associated antigens, such as neoantigens. The methods include culturing of T cells ex vivo in which the T cells have been isolated or obtained from a biological sample as a cellular source for T cells. In some cases, the cellular source includes peripheral blood lymphocytes, lymph node sourced lymphocytes or tumor infiltrating lymphocytes. The methods of culturing the cells include methods to proliferate and expand cells, particularly involving steps to enrich for proliferation and expansion of tumor-reactive T cells such as by selection of such cells or based on T cell activation markers associated with such cells. The provided methods also use certain T cell adjuvants in the ex vivo production of a T cell therapy. In some embodiments, the T cell adjuvant is a costimulatory agonist(s). In some embodiments, the T cell adjuvant is an agent that inhibits apoptosis or an apoptotic pathway in a cell (hereinafter "apoptosis inhibitor"). 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 in the cell. The T cell adjuvant can be added to the manufacturing of T cells to increase the functionality of the T cells ex vivo and for use in in vivo methods of treatment. In particular embodiments, such methods can enrich for expansion of reactive T cells compared to non-reactive and promote their survival and growth in culture ex vivo. It is contemplated that the provided methods can increase expansion to a therapeutic dose to a much greater extent than existing methods and / or increase functionality of the T cell therapy for therapeutic effect. The provided methods can be used to support the growth and survival of donor cells outside of the body, such as in connection with methods of producing a T cell therapy for redelivery back to the patient donor or another patient.

[0147] The provided methods relate to producing a T cell therapy reactive to tumor-associated antigens, such as neoantigens. Cancer cells accumulate many different DNA mutations as part of the tumorigenic process. These mutations can cause amino acid changes in protein coding regions. For a mutation to be recognized by the immune system the protein needs to be processed intracelluarlly and presented the mutant peptide presented on the surface with the Major Histocompatibility Complex (MHC). Peptide neoantigens (also referred to herein as neoepitopes or peptide neoepitopes) are the mutant peptides presented by the MHC complex that can be recognized by a T cell via via TCR binding. In order for the immune system to recognize the mutation, it 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 mutating peptide. These neoantigens may be presented by MHC class I and MHC class II, and are recognized by CD8+ and CD4+ T cells respectively.

[0148] In particular embodiments of the provided methods, the population of T cells is or includes reactive T cells that express cell surface receptors, such as a T cell receptor (TCR), able to recognize peptide antigens on the surface of a target cells. Specifically, for an antigen to be recognized by the immune system the protein needs to be processed intracellularly to peptide fragments that are then presented on the surface with the Major Histocompatibility Complex (MHC). A TCR has two protein chains, which are designed to bind with specific peptides presented by a major histocompatibility complex (MHC) protein on the surface of certain cells. Since TCRs recognize peptides in the context of MHC molecules expressed on the surface of a target cell, 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, such as in tumor, inflammatory and infected microenvironments, and in secondary lymphoid organs. Reactive T cells expressing such cell surface receptors may be used to target and kill any target cell, including, but not limited to, infected cells, damaged cells, or dysfunctional cells. Examples of such target cells may include cancer cells, virally infected cells, bacterially infected cells, dysfunctionally activated inflammatory cells (e.g., inflammatory endothelial cells), and cells involved in dysfunctional immune reactions (e.g., cells involved in autoimmune diseases).

[0149] In some embodiments, a "T cell receptor" or "TCR" is a molecule that contains a variable α and β chains (also known as TCRα and TCRβ, respectively) or a variable γ and δ chains (also known as TCRγ and TCRδ, respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0150] In some aspects, the reactive T cells are tumor-reactive T cells that recognize a cancer antigen. Cancer cells accumulate many different DNA mutations as part of the tumorigenic process. These mutations can cause amino acid changes in protein coding regions. Neoantigens are the mutant peptides encoded by tumor-specific mutated genes and presented by the MHC complex that can be recognized by a T cell via TCR binding. In order for the immune system to recognize the mutation, a neoantigen is expressed on the surface of the cancer cell via the MHC complex for recognition by a T cell that has a TCR that recognizes the mutating peptide. These neoantigens may be presented by MHC class I and MHC class II, and are recognized by CD8+ and CD4+ T cells respectively. The majority of neoantigens arise from passenger mutations, meaning they do not infer any growth advantage to the cancer cell. A smaller number of mutations actively promote tumor grown, these are known as driver mutations. Passenger mutations are likely to give rise to neoantigens that are unique to each patient and may be pressing in a subset of all cancer cells. Driver mutation give rise to neoantigens that are likely to be present in all the tumor cells of an individual and potentially shared. In some embodiments of the provided method, the population of T cells contain tumor-reactive T cells that can recognize neoantigens containing passenger and / or driver mutations.

[0151] In particular aspects, the provided methods can be used for the ex vivo production of a T cell therapy, including for the ex vivo expansion of autologous tumor-reactive T cells. In some aspects, neoantigens are ideal targets for immunotherapies because they represent disease-specific targets. For example, such antigens generally are not present in the body before the cancer developed and are truly cancer specific, not expressed on normal cells and are not subjected to off target immune toxicity. Thus, the unique repertoire of neoantigens specific to the patient can elicit a strong immune response specific to the cancer cells, avoiding normal cells. This is an advantage over other cell therapy targets that may not be disease-specific targets, since even low levels of target antigen on normal cells can lead to severe fatal autoimmune toxicity in the contexts of engineered therapies that target common antigens. For example an anti MAGE-A3-TCR program in melanoma patients was halted due to study related deaths attributed to cross reactivity with a similar target MAGE-A12, which is expressed at a low level in the brain. A significant challenge in cancer immunotherapy has been the identification of cancer targets.

[0152] Recent clinical studies have demonstrated that T cells isolated from surgically resected tumors possess TCRs that recognize neoantigens, and expanding these neoantigen reactive TIL populations and re-infusing them into the patient can in some cases result in a dramatic clinical benefit. This personalized therapy has generated remarkable clinical responses in certain patients with common epithelial tumors.

[0153] Existing methods for obtaining and generating tumor-reactive T cells are not entirely satisfactory. For example, direct isolation of tumor-reactive T cells from a subject without expansion is not feasible because therapeutically effective amounts of such cells cannot be obtained. As an alternative, these cells can be identified through ex vivo co-culture methods of autologous bulk T cells in the presence of autologous antigen-presenting cells. In such methods, autologous antigen-presenting cells are contacted with, or made to present, a source or potential tumor peptides to identify TCRs that are reactive to neoantigen mutations. Although existing methods may result in producing reactive T cells, the procedures often are long, require single cell co-culturing using droplet techniques, and / or involve methods outside of a GMP controlled environment leading to safety risks associated with endotoxins, mycoplasma, and sterility. Attempts also have been made to identify TCRs specific to a desired neoantigen for recombinant engineering of the TCR into T cells for use in adoptive cell therapy methods. Such approaches, however, produce only a single TCR against a specific neoantigen and thereby lack diversity to recognize a broader repertoire of multiple tumor-specific mutations. Other methods involve bulk expansion of T cells from a tumor source, which has the risk of expanding T cells that are not reactive to a tumor antigen and / or that may include a number of bystander cells that could exhibit inhibitory activity. For example, tumor regulatory T cells (Tregs) are a subpopulation of CD4 +< T cells, which specialize in suppressing immune responses and could limit reactivity of a T cell product. These further approaches that have sought to expand tumor-reactive T cells ex vivo are not selective such that non-reactive T cells in the culture may preferentially expand over reactive T cells resulting in a final product that lacks satisfactory reactivity and / or in which the number of tumor-reactive T cells remains insufficient. Methods to produce tumor-reactive T cells for therapy are needed.

[0154] The 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 growth and survival of T cells, such as tumor-reactive T cells, outside of the body. In particular embodiments, the methods enrich for expansion of reactive T cells compared to non-reactive and promote their survival and growth in culture ex vivo. In some embodiments, the resulting methods can be carried out in a closed system. The methods in some embodiments are carried out in an automated or partially automated fashion.

[0155] In provided embodiments, the methods include ex vivo incubation of cells enriched for a population of T cells with a costimulatory agonist under conditions to stimulate or activate a costimulatory receptor expressed by one or more of the T cells in the sample. In particular 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, IL-7, IL-15 and / or IL-21, also can be included during the incubation to initially expand T cells in a population of cells from a subject. In some embodiments, subsequent to or concurrently with incubation with the costimulatory agonist, the population of T cells also is contacted with a T cell stimulatory agent(s), such as 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 to induce or mediate proliferation of T cells in the population. In some embodiments, subsequent to or concurrently with incubation with the costimulatory agent, the population of T cells also is contacted with a T cell stimulatory agent(s), such as an anti-CD3 antibody (e.g. OKT3) or an anti-CD3 / anti-CD28 stimulatory agent, e.g. as anti-CD3 / anti-CD28 beads, under conditions to induce or mediate proliferation of T cells in the population. In some such aspects, the costimulatory agonist, such as a 4-1BB agonist or an OX40 agonist, provides an initial stimulation to enhance or boost the proliferative capacity and / or functional activity of T cells in the population.

[0156] In provided embodiments, the methods include ex vivo incubation of cells enriched for a population of T cells with an apoptosis inhibitor under conditions to reduce or prevent apoptosis of T cells in the sample. In particular embodiments, the apoptosis inhibitor is an inhibitor of the Fas / Fas ligand axis or is an inhibitor of caspase, both of which are involved in inducing apoptosis particularly of activated T cells. In particular embodiments, the apoptosis inhibitor is an inhibitor of one or more caspase (also called caspase inhibitor). As shown herein, caspase inhibitors are found herein to strikingly improve expansion potential of tumor-reactive T cells, particular from a patient tumor or when cells are activated under conditions that may be present in a tumor microenvironment. One or more recombinant cytokines from recombinant IL-2, IL-7, IL-15 and / or IL-21, also can be included during the incubation to initially expand T cells in a population of cells from a subject. In some embodiments, subsequent to or concurrently with incubation with the apoptosis inhibitor, the population of T cells also is contacted with a T cell stimulatory agent(s), such as 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 to induce or mediate proliferation of T cells in the population. In some embodiments, subsequent to or concurrently with incubation with the apoptosis inhibitor, the population of T cells also is contacted with a T cell stimulatory agent(s), such as an anti-CD3 antibody (e.g. OKT3) or an anti-CD3 / anti-CD28 stimulatory agent, e.g. as anti-CD3 / anti-CD28 beads, under conditions to induce or mediate proliferation of T cells in the population. In some such aspects, the apoptosis inhibitor protects the T cells from apoptosis thereby rejuvenating their potential of T cells in the population to proliferate and expand.

[0157] In particular embodiments, the T cell adjuvant, such as a costimulatory agonist, an apoptosis inhibitor, an immune checkpoint modulator, and / or heat shock protein inhibitor. is a soluble protein, such as a protein that is not bound or attached to a solid surface (e.g. a bead or other solid support). The T cell adjuvants can include small molecules, peptides or proteins. Among such T cell adjuvants are soluble ligands, antibody or antigen-binding fragments or other binding agents. In some embodiments, a costimulatory agonist can include a molecule that specifically binds to a costimulatory molecule, such as 4-1BB or OX40, to induce or stimulate a costimulatory signal in the cells. In some embodiments, an apoptosis inhibitor can include a molecule that specifically binds to a receptor that mediates or is involved in inducing apoptosis in a cell. In some embodiments, an immune check point modulator can include a molecule that specficially binds to a "check point" protein , such as PD1. In some embodiments, a heat shock protein inhibitor can include a molecule that specficially binds to a heat shock protein, such as Hsp90. In some embodiments, these molecules can be easily removed during the manufacturing process, such as by washing the cells in connection with cell manufacturing or prior to final formulation of the cells for administration.

[0158] In some embodiments of the provided methods a source of potential tumor peptides is used to identify TCRs that are reactive to neoantigens in a process that includes expansion of the T cells reactive to the tumor neoantigenic peptides. Provided methods include ex vivo co-culture methods in which a population of T cells that have been expanded from a biological sample (e.g. tumor fragments or peripheral blood or other source of T cells) is incubated in the presence of antigen-presenting cells that have been contacted with, or made to present, the neoantigenic peptides. In particular aspects, the T cells and antigen-presenting cells are autologous to the tumor-bearing subject from which the peptides were identified. The provided methods further include steps to separate, enrich for and / or select for tumor-reactive T cells from the co-culture prior to or in connection with their further ex vivo expansion.

[0159] FIG. 1A depicts a schematic of an exemplary process for manufacturing a T cell therapeutic composition in accord with 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-culturing methods with antigen presenting cells (APCs) presenting the peptides and autologous antigen T cells obtained from the same subject. In some cases, a population of T cells from the patient, e.g. containing tumor infiltrating lymphocytes (TIL) or peripheral blood lymphocytes (PBL), is stimulated under conditions to expand the cells, prior to co-culture with antigen presenting cells that have been contacted or exposed to peptide neoepitopes for presentation on a major histocompability complex. Following co-culture under conditions in which the antigen presenting cells present peptides in the context of a major histocompatibility complex, tumor-reactive T cells or T cells surface positive for one or more T cell activation marker (also called an upregulation marker or reactive T cell marker, e.g. CD70a) associated with tumor reactive T cells can be selected and cultured under conditions for expansion in accord with the provided methods, such as incubation with a T cell stimulatory agent(s) (e.g. recombinant IL-2, anti-CD3 and / or anti-CD28). The culturing can be carried out in the presence of one or more recombinant cytokines (e.g. IL-2) to support proliferation and expansion of cells. The process can be carried out in the presence of serum-free media containing nutrients. One or more or all of the steps can be carried out in a closed system, such as without exposure of cells to the environment. Upon reaching a therapeutic dose or a threshold number of cells, the cells can be harvested and formulated, in some cases concentrated or cryopreserved, and used for administration to a subject such as by infusion. In provided examples, one or more of the steps are carried out in the presence of a T cell adjuvant, such as a costimulatory agonist, an apoptosis inhibitor, an immune checkpoint modulator, and / or heat shock protein inhibitor. FIG. 1B depicts an exemplary process in which a cryopreservation step can be carried out after one or more of the steps.

[0160] The provided methods offer advantages compared to existing methods for producing an expanding TILs because the provided methods involve steps to enrich for tumor reactive cells, such as by the co-culturing step with peptide-presenting APCs followed by selection of reactive T cell clones that have upregulated one or more T cell activation marker. By virtue of this process, the initial small population of tumor reactive T cells expanded from the biological sample (e.g. tumor) are enriched for cells that are or likely to be tumor reactive cells before a subsequent second expansion step, thereby promoting preservation and expansion of cells of interest and limiting expansion of bystander T cells that are not reactive to a tumor antigen and / or that may include cells that exhibit inhibitory activity ( FIG. 2A). This is in contrast to existing methods that involve passive expansion of bulk T cells in which all T cells from a tumor are subjected to a first initial expansion, e.g. with high IL-2 concentrations, followed by a second rapid expansion of T cells present after the initial expansion. In such other methods, while total viable cells (TVC) can be greatly expanded by these alternative processes, there is no step of actively ensuring that tumor reactive T cells are predominantly propagated ( FIG. 2B). Further, the provided methods are carried out to maximize the numbers of tumor reactive cells that may be collected, for example by co-culturing all of the cells propagated after the first expansion with peptide-presenting APCs, and then by selecting from among all of the bulk cells after the co-culturing for cells positive for the one or more activation markers before the subsequent second expansion. In aspects of the provided methods, all steps of the method are carried out in a closed system.

[0161] The provided methods include one or more features that provide for or relate to an improved, more efficient and / or more robust process for producing a tumor-reactive T cell therapeutic composition ex vivo. In particular, the disclosure relates to methods that provide advantages over available methods for producing a TIL therapeutic cell composition. 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, including among different subjects and tumor conditions.

[0162] Among the findings herein is the observation that lower concentrations of recombinant IL-2 can be employed during one or both expansion steps with success. Many existing methods use high concentrations of IL-2 of 6000 IU / mL for T cell expansion of TIL. However, high IL-2 concentrations can increase the cost of the process and may be limiting. In some cases, high IL-2 concentrations may lead to negative impacts on T cell differentiation by driving effector T cell differentiation over early memory T cells that may be more desirable in a therapeutic T cell composition. The provided methods can be carried out with concentrations that are several-fold lower than 6000 IU / mL, such as concentrations less than at or about 1000 IU / mL, for example from at or at about 300 IU / mL to at or about 1000 IU / mL. In particular embodiments, the concentration of IL-2 is at or about 300 IU / mL.

[0163] In embodiments of the provided methods, the population of T cells is obtained from a biological sample known to contain T cells. In some embodiments, the population of T cells is enriched from a biological sample from a subject, in particular 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 includes peripheral blood mononuclear cells. In some embodiments, the biological sample is a peripheral blood or 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 is a subject having a cancer, viral infection, bacterial infection, or is a subject with an inflammatory condition. In particular embodiments, the subject has a cancer.

[0164] In aspects of the provided methods, the starting source of cells in the method can be tumor fragments (e.g. 1-8 mm diameter fragments) or can be a single cell suspension preparation from enzymatic digestion of tumor fragments. It is found herein that, while certain sources may be superior for some tumor types, 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 chosen depending on the tumor type or cancer, such as to optimize or increase expansion and enrichment of tumor-reactive T cells from the tumor. In one example, the cancer is a melanoma and the starting population of lymphocytes are tumor fragments, such as from a resected tumor. In another example, the cancer is a colorectal cancer and the starting population of lymphocytes is a single cell suspension obtained by enzymatic digestion, e.g. collagenase, of tumor fragments.

[0165] In some embodiments, the methods include a step of co-culturing initially expanded T cells with autologous antigen presenting cells that have been loaded with peptide. Findings herein demonstrate that relatively low concentrations of peptide or a peptide pool (containing a plurality of peptides, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 more, or any value between any of the foregoing), such where each individual peptide is less than 20 ng / mL, and even as low as 0.1 ng / mL, can lead to an increase in activation of T cell during the culture. In some embodiments, this can lead to an improved enrichment of tumor-reactive T cells in the co-culture prior to selection of cells positive for one or more T cell activation marker (i.e. upregulation marker or reactive T cell marker). In some embodiments, the co-culturing step in the provided methods include a ratio of tumor-derived cells containing T cells to autologous APCs (e.g. dendritic cells) of at or about 1:5 to at or about 5:1, such as 1:3 to at or about 3:1, for example as at or about 1:1, and involves loading the APCs with an individual peptide or a pool of peptides. In some embodiments, the APCs are loaded with a concentration of peptide or peptide pool in which the individual peptide, or individual peptides of the pool of peptides on average, is less than at or about 20 ng / mL, such as from at or about 0.1 ng / mL to at or about 1 ng / mL, for example at or about 0.1 ng / mL.

[0166] In some embodiments, the provided methods include enriching or selecting for a population of T cells from the biological sample. In some aspects, T cells or specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD3+, CD4+ 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+ and CD8+ T cells. For example, CD4+ and CD8+ T cells can be positively selecting for bulk T cells that express CD3. Alternatively, CD4+ and CD8+ T cells can be selected separately, either simultaneously or sequentially in either order, by positive selection of a T cell subpopulation that express CD4 and positive selection of a T cell subpopulation that express CD8. Selection for CD4+ and CD8+ T cells ensures enrichment of T cells expressing MHC class II and MHC class I to provide for a T cell therapy that is a pan-tumor scanning target able to recognize a diverse repertoire of antigens, such as cancer antigens.

[0167] In some embodiments, the provided methods include enriching T cells, such as CD3+ T cells or a CD4 and / or CD8 subset thereof, further based on one or more marker that is whose expression is upregulated on (e.g. compared to resting or non-activated T cells) or specific to reactive or activated T cells (hereinafter "reactive T cell marker" or T cell activation marker). Reactive T cells will express certain reactive markers when their endogenous TCR recognizes an antigen on a target cell or tissue, such as when a TCR recognizes a neoantigen on the tumor. Exemplary reactive T cell markers include one or more, such as 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. The enrichment or selection for cells positive for one or more such reactive T cell marker can be carried out prior to or during one or more steps of the expansion method. In particular embodiments, the provided methods include enrichment or selection for cells positive for one or more upregulation marker on reactive or activated T cells after activation of a population of T cells by the co-culture incubation with peptide-presenting APCs (e.g. dendritic cells, DCs). In some embodiments, the step of selecting cells positive for one or more upregulation marker on reactive or activated T cells from the co-culture can result in 2-fold or greater enrichment of antigen-specific tumor-reactive T cells and / or a substantial decrease in TCR clonality evidencing enrichment of TCR clonotypes consistent with enrichment of tumor-reactive T cells. Furthermore, such enriched T cells can exhibit an improved ability to produce IFN-gamma following antigen-specific stimulation compared to non-selected T cells or bulk T cells from the co-culture.

[0168] In some embodiments, the methods produce or expand T cells for use in adoptive cell therapy methods for treating a disease or condition in which cells or tissue associated with the disease or condition is known or suspected of expressing an antigen 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.

[0169] If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are referenced, the definition set forth herein prevails over the definition that is in the reference.

[0170] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. Ex Vivo Expansion Methods

[0171] Provided herein is a method for the ex vivo expansion and production of a T cell therapeutic composition, particularly for use in connection with treating cancer. In some embodiments, the method of manufacturing involves the growth and manipulation of patient cells outside of the body. In particular embodiments, the methods relate to methods for expanding T cells containing an endogenous TCR specific to a tumor-associated antigen (hereinafter "tumor reactive T cells"). For purposes of this disclosure, reference to tumor reactive T cells includes T cells that exhibit reactivity to a tumor antigen or that are likely or suspected of being tumor reactive T cells due to upregulation or surface positive expression of a T cell activation marker. In some aspects, the frequency of these cells can be low and in order to expand these cells to a therapeutic dose ex vivo methods for enrichment and expansion are necessary.

[0172] Provided methods for expansion of tumor-reactive T cells involve a series of expansion steps to stimulate or induce proliferation of T cells in a population of T cells. In some cases, the methods including incubation 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, in some cases, one or more other stimulatory T cell agents to provide a primary and secondary (costimulatory) signal to the cells. Additionally, in some cases, one or more T cell modulatory agents can be used including apoptosis and heat shock protein inhibitors and immune check point modulators. Standard methods for culturing T cells to provide a primary and secondary (costimulatory) signal to the cells, involve incubation with T cell stimulating agents provided by anti-CD3 (e.g. OKT3) and anti-CD28 reagents. In some embodiments, the T cell stimulatory agents include an anti-CD3 antibody (e.g. OKT3) and an anti-CD28 antibody. Typically such stimulations also include one or more additional recombinant cytokine (e.g. IL-2, IL-7, IL-21 and / or IL-15) and nutrient containing media so that the cells can survive outside of the body.

[0173] In the provided methods, the methods include culturing a population of T cells containing tumor reactive T cells ex vivo in which at least a portion of the culturing includes incubation with additional T adjuvants, including pharmaceutical agonists and in some cases inhibitors of apoptosis or heat shock protein mediated pathways. The addition of one or more T cell adjuvants to the manufacturing of T cells can increase the functionality of the T cells ex vivo and in-vivo. In connection with the provided methods, the methods further include enrichment of T cells containing an endogenous TCR specific to a tumor-associated antigen ("tumor reactive T cells") to maximize expansion of desired therapeutic cells. In some embodiments, the tumor-associated antigen is or includes a neoantigen.

[0174] Thus, among the provided methods are methods of culturing T cells for manufacture of tumor reactive T cells that involve both (1) the use of additional T cell modulatory agents, such as prior to or concurrently with standard T cell stimulatory agent(s) such as anti-CD3 / anti-CD28 and / or recombinant cytokines (e.g. IL-2, IL-7, IL-21 and / or IL-15), and (2) further involve enrichment 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. It is contemplated that the provided methods can increase expansion to a therapeutic dose to a much greater extent than existing methods and / or increase functionality of the T cell therapy for therapeutic effect.

[0175] The provided methods involve collecting a biological sample from a subject that is known or likely to contain tumor reactive T cells. In some embodiments, the biological sample can be collected directly from a subject that has a tumor, in which, in some cases, such isolated or obtained T cells may have been co-cultured or exposed to a tumor in vivo. In embodiments of provided methods, a population containing T cells (hereinafter also called first population of T cells) is a population of T cells obtained, selected or isolated from the biological sample from a subject, such as a human subject. In some embodiments, the population containing T cells can be from any source sample that is known or suspected of containing T cells that are or that may include or potentially could include tumor reactive T cells. The sample can include a tumor sample containing tumor infiltrating lymphocytes (TILs), a blood sample (e.g. apheresis or leukapheresis sample) containing peripheral blood mononuclear cells (PBMCs) or a lymph node sample. In some embodiments, the sample is a tumor sample or a tumor fragment containing tumor infiltrating lymphocytes or TILs. The population containing T cells can be directly obtained from a subject (e.g. healthy or cancer subject), such as by selection of T cells or a subset thereof from the biological sample from the subject. In particular embodiments, the biological sample is from a subject that has a tumor and that contains tumor reactive T cells or that has the potential to or that may contain tumor reactive T cells that can be enriched by the provided methods.

[0176] Provided methods for expansion of tumor-reactive T cells involve a first expansion involving culturing the selected or isolated population containing T cells (i.e. the first population of T cells) with a recombinant cytokine from one or more of (e.g. IL-2, IL-7, IL-21 and / or IL-15), typically generally including recombinant IL-2. In some cases, the T cell stimulatory agent(s) tfurther provide a primary and secondary (costimulatory) signal to the cells, such as provided by anti-CD3 (e.g. OKT3) and anti-CD28 reagents, such as an anti-CD3 antibody (e.g. OKT3) and an anti-CD28 antibody.. In some cases, the first expansion also is carried out in the presence of one or more T cell modulatory agent, e.g. TNFSFR agonists and / or immune checkpoint modulators and / or apoptosis inhibitors and / or heat shock protein inhibitors as described. The initial or first expansion results in a second population of T cells that is enriched for T cells as a result of expansion or proliferation of T cells present in the first population.

[0177] In other embodiments of the provided methods, the biological sample can be collected and used for ex vivo co-culture in which T cells directly obtained from a subject (e.g. healthy or cancer subject) have been incubated with artificial presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject. The collected samples contain lymphocytes that have endogenous TCRs that are reactive to mutations present on the tumor. These cells can be identified via a variety of methods such as, but not limited to, co-culture in the presence of antigen presenting cells or co-culture with 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 further steps that include ex vivo co-culture of the stimulated or expanded T cells (i.e. second population of T cells) with antigen presenting cells and one or a plurality of peptides that include neoepitopes of a tumor antigen (APCs / peptide neoepitopes). Provided methods include ex vivo co-culture in which the second population of T cells are incubated with artificial presenting cells (APCs) that have been exposed to or contacted with one or more peptides, e.g. synthetic peptides, under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen. In some embodiments, the population T cells are autologous T cells from a subject with a tumor and the source of synthetic peptides are tumor antigenic peptides from a tumor antigen of the same subject. In some embodiments, cells from the ex vivo co-culture include population of T cells (in some cases a third population of T cells) that represent a source of cells that are enriched for tumor reactive T cells. In some cases, the co-culture of T cells with APCs and peptides can also be carried out in the presence of one or more T cell modulatory agent (e.g. TNFSFR agonist and / or apoptosis inhibitor).

[0178] In some cases, the tumor reactive T cells can be further enriched by separation or selection of cells that express one or more activation markers associated with tumor-reactive T cells. Among the provided aspects, a biological sample containing tumor-reactive T cells, including a sample obtained directly from a subject or a co-culture sample generated ex vivo, 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. The T cell activation markers include cell surface markers whose expression is upregulated or specific to T cells that have been exposed to antigen and activated. Exemplary of such markers are described below. In provided aspects, a population of T cells containing tumor-reactive T cells is isolated, selected or enriched from a biological sample prior to or in connection with expanding such cells in accord with the provided methods. In some embodiments, the tumor-reactive T cells, or T cells that express certain activation markers associated with tumor-reactive T cells, are those present endogenously in a cancer subject known to have a tumor. In other embodiments, the tumor-reactive T cells, or T cells that express certain activation markers associated with tumor-reactive T cells, are obtained following an ex vivo co-culture of autologous T cells from a subject with a tumor with a source of tumor antigenic peptides under conditions in which tumor-reactive T cell can be generated and identified.

[0179] In particular embodiments, a second expansion is performed on T cells enriched or isolated from the co-culture, 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 T cells with a T cell stimulatory agent(s), such as anti-CD3 antibody (e.g. OKT3), anti-CD28 antibody, and recombinant cytokine(s) (e.g. IL-2, IL-7, IL-21 and / or IL-15), and optionally one or more T cell modulatory agent (e.g. costimulatory agonists and / or immune checkpoint modulators and / or apoptosis inhibitors and / or heat shock protein inhibitors as described). The T cells, such as 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, are allowed to expand for a certain number of days as desired and / or until a therapeutic dose or harvest dose is met. The composition of expanded T cells can then be harvested and formulated for administration to a subject for treatment of a cancer in the subject.

[0180] Provided herein are methods for manufacturing tumor-reactive T cells in which cells are cultured ex vivo by a process that includes incubating a population of T cells containing, or that is likely or may contain, tumor-reactive T cells, said tumor-reactive T cells comprising an endogenous TCR that is 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 at least a portion of the incubation is carried out prior to, concurrently with, or subsequent to incubating the population of T cells with a T cell stimulatory agent(s), e.g. anti-CD3 antibody (e.g. OKT3), anti-CD28 antibody and / or a recombinant cytokine(s) (e.g.from IL-2, IL-7, IL-21 and / or IL-15), under conditions to stimulate expansion of cells of the population of T cells. The T cell adjuvant, e.g. costimulatory agonists and / or immune checkpoint modulators and / or apoptosis inhibitors and / or heat shock protein inhibitors as described, can be included during either one or both of the first expansion or the second expansion in accord with the provided methods. In some embodiments, the methods of culturing includes (a) incubating an ex vivo population of T cells comprising tumor-reactive T cells, said tumor-reactive T cells comprising an endogenous TCR that is 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 population of T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of cells of the population of cells, wherein at least a portion of the incubating to stimulate or expand the T cells, e.g. in either one or both of the first or second expansion, is carried out prior to, concurrently with, or subsequent to the incubating with the at least one T cell adjuvant. In some embodiments, the population of T cells that is incubated in the presence of the at least one T cell adjuvant can be the first population of T cells following obtaining, selecting or isolating the population of T cells from a biological sample from a subject. In embodiments, the population of T cells is enriched for CD4+ and CD8+ T cells. In some cases, tumor-reactive T cells can be enriched from a biological sample comprising T cells prior to the incubating with the at least one T cell adjuvant and / or prior to the incubating with the at least one T cell stimulatory agent(s). In some embodiments, the population of T cells that is incubated in the presence of the at least one T cell adjuvant can be the population of T cells that has been enriched for or selected for tumor reactive T cells or T cells that express an activation marker following co-culture with APCs / peptide neoepitopes (e.g. in some cases the fourth population of 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 until up to 20 days after initiation of incubation with the at least one T cell adjuvant, wherein at least a portion of the culturing is carried out during the incubation with the one or more T cell adjuvant and / or the incubation with the T cell stimulatory agent(s).

[0181] Provided herein are methods for manufacturing tumor-reactive T cells in which cells are cultured ex vivo by a process that includes a step of enriching tumor-reactive T cells from a sample comprising T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and incubating the population of T cells enriched for 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 is carried out prior to, concurrently with, or subsequent to incubating the population of T cells with a T cell stimulatory agent(s), e.g. anti-CD3 antibody (e.g. OKT3), anti-CD28 antibody and / or recombinant cytokine(s) (e.g. IL-2, IL-7, IL-21 and / or IL-15), under conditions to stimulate expansion of cells of the population of T cells. In some embodiments, the tumor-reactive T cells are selected or isolated after co-culturing a population of T cells containing stimulated T cells (in some cases the second population of 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 peptide. In some embodiments, the methods of culturing include (a) enriching tumor-reactive T cells from a biological sample comprising T cells, such as from a co-culture of T cells and APCs / neoepitope peptides, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; (b) incubating the population of T cells enriched for tumor-reactive T cells with at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor; and (c) incubating the population of T cells enriched for tumor-reactive T cells with a T cell stimulatory agent(s) under conditions to stimulate expansion of cells of cells of the population of cells, wherein at least a portion of the incubating with the T cell stimulatory agent is carried out prior to, concurrently with, or subsequent to the incubating with the at least one T cell adjuvant. In embodiments, the population of T cells is enriched for CD4+ 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 until up to 20 days after initiation of incubation with the at least one T cell adjuvant, wherein at least a portion of the culturing is carried out during the incubation with the one or more T cell adjuvant and / or the incubation with the T cell stimulatory agent(s).

[0182] Provided herein is a method for manufacturing tumor reactive T cells in which cells are cultured ex vivo by a process that includes incubating a population of T cells with a T cell stimulatory agent(s), e.g. arecombinant cytokine(s) from amongIL-2, IL-7, IL-21 and / or IL-15, such as generally at least including recombinant IL-2, and at least one T cell adjuvant selected from a costimulatory agonist or an apoptosis inhibitor, in which the incubation is carried out under conditions to stimulate or expand T cells in the population to produce a second population of T cells; enriching tumor-reactive T cells from the population of T cells, said tumor-reactive T cells comprising an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and incubating the population of T cells enriched for tumor-reactive T cells with a T cell stimulatory agent(s), e.g. anti-CD3 antibody (e.g. OKT3), anti-CD28 antibody and recombinant cytokine(s) (e.g. IL-2, IL-7, IL-21 and / or IL-15), under conditions to further stimulate expansion of cells of the population of cells enriched in tumor reactive T cells. In some embodiments, the tumor-reactive T cells are selected or isolated after co-culturing a population of T cells containing stimulated T cells (in some cases the second population of 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 peptide. In embodiments, the population of T cells is enriched for CD4+ 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 until up to 20 days after initiation of incubation with the at least one T cell adjuvant, wherein at least a portion of the culturing is carried out during the incubation with the one or more T cell adjuvant and / or the incubation with the T cell stimulatory agent(s).

[0183] In particular embodiments, the provided methods include, but are not limited to the steps of (1) identifying, obtaining or generating a plurality of peptides that contain neoepitopes specific to a subject's tumor (2) obtaining a population of T cells obtained from a donor subject, such as from a resected tumor or by directly selecting T cells from a biological sample, e.g. a tumor, blood, bone marrow, lymph node, thymus or other tissue or fluids (first population of T cells); (3) performing a first expansion by stimulating or activating the first population of T cells with a T cell stimulatory agent(s), such as one or more recombinant cytokines from IL-2, IL-7, IL-21 and / or IL-15 (e.g. at least including recombinant IL-2), and optionally one or more further T cell modulatory agent, such as a TNFRSF agonist and / or apoptosis inhibitor to produce a second population of T cells containing expanded or stimulated T cells, (3) co-culturing the second population containing 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 peptide to produce a third population of T cells; and (5) enriching, from the third population of T cells, T cells containing an endogenous TCR that are reactive to peptides present on antigen presenting cells (APCs) to produce a fourth population of T cells. In some aspects, T cells containing an endogenous TCR are enriched by separating the antigen presenting cells from the population of T cells. 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 particular embodiments, a second expansion is performed on T cells enriched or isolated from the co-culture, 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 T cells with a T cell stimulatory agent(s), such as anti-CD3 antibody (e.g. OKT3), anti-CD28 antibody and recombinant cytokine(s) (e.g. IL-2, IL-7, IL-21 and / or IL-15), and optionally one or more T cell modulatory agent (e.g. TNFSFR agonists and / or immune checkpoint modulators and / or apoptosis inhibitors and / or heat shock protein inhibitors ).

[0184] In embodiments of the provided methods, one or more of the steps can be carried out in serum-free media. 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, e.g., up to 5%, e.g., about 1%, 2%, 3%, 4%, or 5%. In some embodiments, the serum-free media contains 0.5 mM to 5 mM of a dipeptide form of L-glutamine, such L-alanyl-L-glutamine (Glutamax ™< ). In some embodiments, the concentration of the dipeptide form of L-glutamine, such as L-alanyl-L-glutamine, is from or from about 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 4 mM to 5 mM, each inclusive. In some embodiments, the concentration of the dipeptide form of L-glutamine, such as L-alanyl-L-glutamine, is or is about 2 mM.

[0185] In embodiments of any of the provided methods, the incubation with each of the at least one T cell adjuvant, such as one or more costimulatory agonist, immune checkpoint modulator, heat shock protein inhibitor, or apoptosis inhibitor, is independently continued during the entire course of the culturing or during a portion thereof. In some embodiments, the incubation with each of the at least one T cell adjuvant is for no more than 14 days, no more than 12 days, no more than 10 days, no more than 7 days, no more than 5 days, no more than 3 days or no more than 2 days. In some embodiments, the incubation with each of the at least one T cell adjuvant is independently for 12 hours to 96 hours, such as 24 hours to 48 hours, and generally is at or about 48 hours.

[0186] In embodiments, the incubation with each of the T cell stimulatory agent(s), such as a recombinant cytokine (e.g. IL-2) and / or an anti-CD3 / anti-CD28 antibody, can be continued for a time period sufficient to activate or stimulate the cells. In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out for at or about 1 day, such as generally at or about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or any range of time between any of the foregoing. In some embodiments, the incubation is carried out for 7-10 days. In some embodiments, the incubation is for at or about 7 days. In some embodiments, the incubation is for at or about 8 days. In some embodiments, the incubation is for at or about 9 days. In some embodiments, the incubation is for at or about 10 days. In some embodiments, the incubation with each of the T cell stimulatory agent(s), such as a recombinant cytokine (e.g. IL-2) and / or anti-CD3 / anti-CD28 antibody, is for 12 hours to 96 hours, such as 24 hours to 48 hours, and generally at or about 48 hours.

[0187] In some embodiments, the cells are washed one or more times during the culturing to remove agents present during the culturing and / or to replenish the culture medium with one or more additional agents. In some embodiments, the cells are washed during the cultureing to reduce or remove the at least one T cell adjuvant and / or at least one T cell stimulatory agent(s) prior to completion of the culturing.

[0188] In some embodiments, the methods of culturing T cells provided herein, including incubation with a T cell adjuvant and / or a T cell stimulatory agent(s), include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. In some embodiments, the methods of culturing, including incubation with a T cell adjuvant and / or a T cell stimulatory agent(s) is carried out in serum-free media.

[0189] In particular embodiments, the provided methods include enriching from a biological sample (directly sourced from a sample in vivo or from an ex vivo co-culture with antigen presenting cells (APCs) T cells that have an endogenous TCR that recognize tumor-associated antigens, e.g. neoantigens, such as by selecting for T cells that are surface positive for one or more T cell activation marker (e.g. CD107, CD107a, CD039, CD137, CD59, CD90, CD38, or CD103).

[0190] In some embodiments, any one or more of the steps of the method can be carried out 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 for carrying out one or more of the other processing steps of a method for manufacturing, generating or producing a cell therapy. In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and / or enrichment, processing, culturing steps including incubation in connection with expansion of the cells, and formulation steps is carried out using a system, device, or apparatus in an integrated or self-contained system, and / or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0191] In some embodiments, the methods of culturing for expanding cells in accord with any of the provided methods is carried out until a threshold amount of cells, such as tumor-reactive cells or cells positive for one or more T cell activation marker, is obtained and / or until up to 20 days after initiation of the incubation with the at least one T cell adjuvant. In some embodiments, the culturing is carried out for 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.

[0192] In some embodiments, the culturing is carried out until a threshold amount of cells is obtained in which the threshold amount is between at or about 0.5 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between 0.5 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 8 x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 3.5x 10 8< total cells or total viable cells, between at or about 0.5 x 10 8< and at or about 1 x 10 8< total cells or total viable cells, between 1 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 1 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between 1 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 1 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 1 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 1 x 10 8< and at or about 8 x 10 8< total cells or total viable cells, between at or about 1 x 10 8< and at or about 3.5x 10 8< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 3.5 x 10 8< and at or about 8 x 10 8< total cells or total viable cells, between at or about 8 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 8 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 8 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 8 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 8 x 10 8< and at or about 15 x 10 8< total cells or total viable cells, between at or about 15 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 15 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 15 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 15 x 10 8< and at or about 60 x 10 8< total cells or total viable cells, between at or about 60 x 10 8< and at or about 50 x 10 9< total cells or total viable cells, between at or about 60 x 10 8< and at or about 30 x 10 9< total cells or total viable cells, between at or about 60 x 10 8< and at or about 12 x 10 9< total cells or total viable cells, between at or about 12 x 10 9< and at or about 50 x 10 9< total cells or total viable cells, between at or about 12 x 10 9< and at or about 30 x 10 9< total cells or total viable cells, or between at or about 30 x 10 9< and at or about 60 x 10 9< total cells or total viable cells, each inclusive.

[0193] In some of any of the provided embodiments, the method results in a fold-expansion of T cells or in a fold-expansion of tumor reactive T cells that is at least at or about 2-fold, at least at or about 5-fold, at least at or about 10-fold, at least at or about 25-fold, at least at or about 50-fold, at least at or about 100-fold, at least at or about 250-fold, at least at or about 500-fold, at least at or about 1000-fold, or more.

[0194] Non-limiting descriptions of aspects of the provided methods are further described in the following subsections.A. Neoepitope Identification and Peptide Generation

[0195] The provided methods include a step of generating or identifying in silico a plurality of peptides (also referred to as "P" or "n-mers") that contain at least one cancer-specific cancer neoepitope, and a further step of filtering in silico the peptides to so 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 employed in methods to enrich for tumor-reactive T cells in accord with the provided methods.

[0196] In some embodiments, methods for ex-vivo generation of tumor-reactive T cells include identifying or isolating a tumor-associated antigen or peptide sequence thereof from a cancer cell from a subject. The cancer cell may be obtained from any bodily sample derived from a patient which contains or is expected to contain tumor or cancer cells. The bodily sample may be any tissue sample such as blood, a tissue sample obtained from the primary tumor or from tumor metastases, a lymph node sample or any other sample containing tumor or cancer cells. In some aspects, nucleic acid from such cancer cells is obtained and sequenced. In embodiments, the protein-coding region of genes in a genome is sequenced, such as by whole exome sequencing. To identify tumor-specific sequences, sequencing data can be compared to a reference sequencing data, such as data obtaining by sequencing a normal cell or noncancerous cell from the same subject. In some embodiments, next-generation sequencing (NGS) methods are used.

[0197] In some embodiments, the tumor is a hematological tumor. Non- limiting examples of hematological tumors include leukemias, including acute leukemias (such as l lq23- positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias 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 high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0198] 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 carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, 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, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). In several examples, a tumor is melanoma, lung cancer, lymphoma breast cancer or colon cancer.

[0199] In some embodiments, the cancer is a gastrointestinal cancer involving a cancer of the gastrointestinal tract (GI tract), including cancers or the upper or lower digestive tract, or an accessory organ of digestion, such as esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum or anus. In some embodiments, the cancer is an espphageal cancer, stomach (gastric) cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, cancer of the mucosa-associated lymphoid tissue (MALT lymphoma), cancer of the biliary tree, colorectal cancer (including colon cancer, rectum cancer or both), anal cancer, or a gastrointestinal carcinoid tumor. In particular embodiments, the cancer is a colorectal cancer.

[0200] In some embodiments, the tumor is from a breast cancer, such as a ductal carcinoma or a lobular carcinoma. In some embodiments, the tumor is from a prostate cancer. In some embodiments, tumor is from a skin cancer, such as a basal cell carcinoma, a squamous cell carcinoma, a Kaposi's sarcoma, or a melanoma. In some embodiments, the tumor is from a lung cancer, such as an adenocarcinoma, a bronchiolaveolar carcinoma, a large cell carcinoma, or a small cell carcinoma. In some embodiments, the tumor is from a brain cancer, such as a glioblastoma or a meningioma. In some embodiments, the tumor is from a gastrointestinal cancer, such as any described above. In some embodiments, the tumor is from a colon cancer. In some embodiments, the tumor is from a liver cancer, such as a hepatocellular carcinoma. In some embodiments, the tumor is from a pancreatic cancer. In some embodiments, the tumor is from a kidney cancer, such as a renal cell carcinoma. In some embodiments, the tumor is from a testicular cancer.

[0201] In some embodiments, the cancer is not a melanoma. Melanoma is a cancer that generally has a high mutational rate. High tumor mutation burden has been thought to be a particularly desired prognostic marker for success related to treatment with an immunotherapy 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 methods can be used in cancers that have a lower tumor mutation burden, since the methods are carried out to actively (as opposed to passively) enrich for tumor reactive T cells.

[0202] In some embodiments, the subject is a subject with a tumor mutational burden (TMB) of less than 8 mutations.TMB includes the number of non-synomymous mutations per tumor. In some embodiments, TMB can be calculated by counting the number of synonymous and non-synonymous mutations across a 0.8- to 1.2-megabase (Mb) region, and reporting the result as mutations / Mb. In some embodiments, TMB can be determined by next generation sequencing (NGS) on 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 at or about 60 mutations / Mb, such as less than at or about 55 mutations / Mb, less than at or about 50 mutations / Mb, less than at or about 45 mutations / Mb, less than at or about 40 mutations / Mb, less then at or about 30 mutations / Mb, less than at or about 25 mutations per Mb, or less than at or about 20 mutations / Mb, or any value between any of the foregoing. In some embodiments, the subject has a TMB of less than at or about 41 mutations / Mb, less than at or about 40 mutations / Mb, less than at or about 39 mutations / Mb, less than at or about 38 mutations / Mb, less than at or about 37 mutations / Mb or less.

[0203] In some embodiments, the peptide (P) is a tumor-associated antigen derived from premalignant conditions, such as variants of carcinoma in situ, or vulvar intraepithelial neoplasia, cervical intraepithelial neoplasia, or vaginal intraepithelial neoplasia.

[0204] In some aspects, nucleic acid from such cells of the tumor or cancer is obtained and sequenced. In embodiments, the protein-coding region of genes in a genome is obtained, such as by omics analysis, such as by analysis of whole genomic sequencing data, exome sequencing data, and / or transcriptome data. To identify tumor-specific sequences, sequencing data can be compared to a reference sequencing data, such as data obtaining from a normal cell or noncancerous cell from the same subject. In some embodiments, next-generation sequencing (NGS) methods are used.

[0205] In some embodiments, the methods include a step of using matched normal omics data of a tumor. In such methods, the in silico analysis involves an omics analysis to identify mutations in the tumor relative to normal tissue of the same patient, such as non-diseased tissue of the same patient. It is generally contemplated that matched normal omics data are whole genomic sequencing data, exome sequencing data, and / or transcriptome data, and that the matched normal omics data are matched against normal before treatment of the patient. In a particular embodiment, whole exome sequencing is performed on healthy and diseased tissue to identify somatic mutations associated with the tumor.

[0206] In some embodiments, omics data are obtained from one or more patient biopsy samples following standard tissue processing protocol and sequencing protocols. In particular embodiments, the data are patient matched tumor data (e.g., tumor versus same patient normal). In some cases, non- matched or matched versus other reference (e.g., prior same patient normal or prior same patient tumor, or homo statisticus) are also deemed suitable for use herein. The omics data may be fresh omics data or omics data that were obtained from a prior procedure (or even different patient). For example, neoepitopes may be identified from a patient tumor in a first step by whole genome and / or exome analysis of a tumor biopsy (or lymph biopsy or biopsy of a metastatic site) and matched normal tissue (i.e., non-diseased tissue from the same patient such as peripheral blood). In some embodiments, genomic analysis can be processed via location-guided synchronous comparison of the so obtained omics information.

[0207] The genomic analysis can be performed by any number of analytic methods. In particular embodiments, the methods include WGS (whole genome sequencing) and exome sequencing of both tumor and matched normal sample using next generation sequencing such as massively parallel sequencing methods, ion torrent sequencing, pyrosequencing. Computational analysis of the sequence data may be performed in numerous manners. In some embodiments, the data format is in SAM, BAM, GAR, or VCF format. As an example, analysis can be performed in silico by location-guided synchronous alignment of tumor and normal samples as, for example, disclosed in US 2012 / 0059670A1 and US 2012 / 0066001 Al using BAM files and BAM servers. Alternative file formats for sequence analysis (e.g., SAM, GAR, FASTA, etc.) are also contemplated.

[0208] In some of any embodiments, peptides (P) comprising neoantigens arising from a missense mutation encompass the amino acid change encoded by 1 or more nucleotide polymorphisms. Peptides (P) comprising neoantigens that arise from frameshift mutations, splice site variants, insertions, inversions and deletions should encompass the novel peptide sequences and junctions of novel peptide sequences. Peptides (P) comprising neoantigens with novel post-translational modifications should encompass the amino acids bearing the post-translational modification(s), such as a phosphate or glycan.

[0209] Once these mutations are identified, neoepitopes are then identified. Neoepitopes are mutant peptides that are recognized by a patient's T cells. These neoepitopes must be presented by a tumor or antigen presenting cell by the MHC complex and then be recognized by a TCR on the T cell. In some embodiments, the provided methods include a step of calculation of one or more neoepitopes to define neoepitopes that are specific to the tumor and patient. Consequently, it should be recognized that patient and cancer specific neoepitopes can be identified from omics information in an exclusively in silico environment that ultimately predicts potential epitopes that are unique to the patient and tumor type. In particular aspects, the so identified cancer neoepitopes are unique to the patient and the particular cancer in the patient (e.g., having a frequency of less than 0.1% of all neoepitopes, and more typically less than 0.01% in a population of cancer patients diagnosed with the same cancer), but that the so identified cancer neoepitopes have a high likelihood of being presented in a tumor.

[0210] In some of any embodiments, the length of the peptide (P) depends on the specific application and is typically between about 5 to about 50 amino acids. In preferred embodiments, the peptide (P) is between 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 methods can be carried out with an individual peptide that includes a change(s) (e.g. mutations) in the amino acid sequences. In some aspects, the methods can be carried out with a pool of peptides, where peptides of the pool contain a change(s) (e.g. mutations) in the amino acid sequences. The pool of peptides can include tens to hundreds of individual peptides. In some cases, the pool of peptides includes 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. The pool of peptides can represent one neo-antigen or can represent several neoantigens. In some cases, a pool of peptides can include multiple overlapping peptides of the same neo-antigen. Thus, for a tumor-associated antigen, the antigen may be divided into 7 to 35 amino acid, e.g., 25 amino acid, peptides (P) wherein each peptide (P) contains a unique composition of amino acids; or, the peptides (P) can be overlapping peptide pools wherein an antigen is divided into a set number of 7 to 35 amino acid, e.g., 25 amino acid, peptides (P) that have overlapping sequences. For example, an overlapping peptide pool comprising a 100 amino acid antigen may be divided into eight 25 amino acid peptides (P) that are each offset by 12 amino acids (i.e., each subsequent 25 amino acid peptide comprising a 100 amino acid peptide sequence starts at the 13 th< amino acid position from the prior peptide). Those skilled in the art understand that many permutations exist for generating a peptide pool from an antigen.

[0211] The neoepitope sequences as contemplated herein can be defined as sequence stretches with relatively short length (e.g., 5-30 mers, more typically 7- 11 mers, or 12-25 mers) wherein such stretches include the change(s) (e.g. mutations) in the amino acid sequences. Most typically, the change(s) is / are located centrally or near the center (e.g., less than 4, or less than 5, or less than 6 amino acids from center position). In particular aspects, neoepitope sequences contemplated herein will especially include those in which a single amino acid is exchanged relative to the matched normal sequence, and in which the position of the changed amino acid is centrally located, 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, and more typically at position 3, 4, or 5, and most typically at position 4 or 5). It should be appreciated that a single amino acid change may be presented in numerous neoepitope sequences that include the changed amino acid, depending on the position of the changed amino acid.

[0212] In particular embodiments, neoepitopes will be calculated to have a length of between 2-50 amino acids, more typically between 5-30 amino acids, and most typically between 9- 15 amino acids. For example, where the epitope is to be presented by the MHC-I complex, a typical epitope length will be about 8- 11 amino acids, while the typical epitope length for presentation via MHC-II complex will have a length of about 13- 17 amino acids. As will be readily appreciated, since the position of the changed amino acid in the neoepitope may be other than central, the actual peptide sequence and with that actual topology of the neoepitope may vary considerably. Moreover, where the neoepitope is presented to an immune competent (or other) cell as a synthetic peptide, it should be appreciated that the synthetic peptide may be significantly longer than the peptide portion that is ultimately bound by the MHC-I or MHC-II system to so allow for proteolytic processing in the cell. For example, contemplated synthetic peptides may therefore have between 8 and 15 amino acids upstream and downstream of the changed amino acid.

[0213] Various algorithms have been developed and can be used to map T cell epitopes (both MHC Class I and Class II-restricted) within protein molecules of various origins. In some embodiments, many programs utilize availability of the large-scale peptide-MHC binding affinity matrix 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 predictor methods for MHC class I (e.g. 9-mer) include smm, smmpmbec, ann (NetMHC3.4), NetMHC4, PickPocket, consensus, NetMHCpan2.8, NetMHCpan3, NetMHCpan4, NetMHCcons, mhcflurry, mheflurry_pan, or MixMHCpred. Exemplary predictor methods for MHC class II (e.g. 15-mer ) include NetMHCIIpan, NetMHCII2.3, nn_align, smm_align, consensus, comblib, tepitope, or mhcflurry. Any of such methods can be used.

[0214] In embodiments where the synthetic peptide is used for direct MHC-I binding, the overall length will be between 8 and 10 amino acids. In embodiments, where the synthetic peptide is used for direct MHC-II binding, the overall length will be between 12 and 25 amino acids, such as between 14 and 20 amino acids. In some cases, where the synthetic peptide is processed in the cell (typically via proteasome processing) prior to MHC presentation, the overall length will typically be between 10 and 40 amino acids, with the changed amino at or near a central position in the synthetic peptide. In some embodiments, a peptide for MHC-I binding is a 9-mer. In some embodiments, a peptide for MHC-II binding is a 23-mer. In some embodiments, a peptide for MHC-II binding is a 25-mer.

[0215] As an example, a peptide (P) can include 0-25 amino acids on either side flanking the amino acid change or novel junction that arises due to a mutation. In one embodiment, the peptide (P) is a neoantigen sequence that comprises the 12 amino acids on either side flanking the amino acid change that arises from a single nucleotide polymorphism, for example, a 25 amino acid peptide, wherein the 13 th< amino acid is the amino acid residue resulting from the single nucleotide polymorphism. In some embodiments, the peptide (P) is a neoantigen sequence that comprises the 12 amino acids on either side flanking an amino acid with a novel post-translational modification, for example, a 25 amino acid peptide, wherein the 13 th< amino acid is the amino acid residue resulting from the novel post- translational modification site. In other embodiments, the peptide (P) is a neoantigen sequence that comprises 0-12 amino acids on either side flanking a novel junction created by an insertion, deletion or inversion. In some cases, the peptide (P) comprising neoantigens resulting from novel sequences can encompass the entire novel sequence, including 0-25 amino acids on either side of novel junctions that may also arise.

[0216] In some embodiments, further downstream analysis may be performed on the so identified sequence differences to identify those that lead to a new peptide sequence based on the cancer and patient specific mutation. Neoepitopes may therefore be identified by considering the type (e.g., deletion, insertion, transversion, transition, translocation) and impact of the mutation (e.g., non-sense, missense, frame shift, etc.), and may as such serve as a content filter through which silent and other non-relevant (e.g., non-expressed) mutations are eliminated.

[0217] In some embodiments, identified neoepitopes can be further filtered in silico against an identified patient HLA- type. Such HLA-matching is thought 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 presentation systems is particularly thought to produce a therapeutically effective and durable immune response involving both, the cellular and the humoral branch of the immune system. It should also be appreciated that thusly identified HLA-matched neoepitopes can be biochemically validated in vitro.

[0218] HLA determination for both MHC-I and MHC-II can be done using various methods. In some embodiments, the HLA-type can be predicted from omics data in silico using a reference sequence containing most or all of the known and / or common HLA-types. For example, a patient' s HLA-type is ascertained (using wet chemistry or in silico determination), and a structural solution for the HLA-type is calculated or obtained from a database, which is then used as a docking model in silico to determine binding affinity of the neoepitope to the HLA structural solution. Suitable systems for determination of binding affinities include the NetMHC platform (see e.g., Nucleic Acids Res. 2008 Jul 1; 36(Web Server issue): W509-W512.), HLAMatchmaker (http: / / www.epitopes.net / downloads.html), and IEDB Analysis Resource (http: / / tools.immuneepitope.org / mhcii / ). Neoepitopes with high affinity (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) against the previously determined HLA-type are then selected. In calculating the highest affinity, modifications to the neoepitopes may be implemented by adding N- and / or C-terminal modifications to the epitope to further increase binding of a synthetic neoepitope to the HLA-type of the patient. Thus, neoepitopes may be native as identified or further modified to better match a particular HLA-type. In some embodiments, neoepitopes can be scored / ranked based on allele frequency multiplied by the transcripts per million number to get a likelihood score. This score can then be further augmented using HLA information and calculated or actual binding affinity to the patient' s HLA type.

[0219] Among provided embodiments are embodiments in which the neoepitopes are compared against a database that contains known human sequences to so avoid use of a human-identical sequence.

[0220] After the in silico identification of suitable neoepitope sequences, corresponding synthetic peptides are then prepared in vitro (e.g. , using solid phase synthesis). In particular embodiments, a library of synthetic peptides is prepared representing a plurality of different neoepitopes from the subject. The library can include 100, 1000, 10000 or more different peptides. To obtain a synthetic antibody against the identified neoepitope(s), it is contemplated that the in silico identified is prepared in vitro to yield a synthetic peptide.

[0221] Various methods can be used to prepare synthetic peptides. For example, peptides with cancer neoepitope sequences can be prepared on a solid phase (e.g., using Merrified synthesis), via liquid phase synthesis, or from smaller peptide fragments. Peptide epitopes can be obtained by chemical synthesis using a commercially available automated peptide synthesizer. In some embodiments, the peptides can be synthesized, for example, by using the Fmoc-polyamide mode of solid-phase peptide synthesis which is disclosed by Lu et al (1981).J. Org. Chem. 46,3433 and the references therein. In some aspects, peptides can be produced by expression of a recombinant nucleic acid in a suitable host and with a suitable expression system. In some aspects, recombinant methods can be used where multiple neoepitopes are on a single peptide chain, such as with spacers between neoepitopes or cleavage sites).

[0222] The 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 reverse-phase high performance liquid chromatography using e.g. acetonitrile / water gradient separation. In some embodiments, peptides can be precipitated and further purified, for example by high performance liquid chromatography (HPLC). Analysis of peptides can be carried out using thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF mass spectrometric analysis.B. Cell Samples Containing Tumor-Reactive T cells

[0223] The provided methods include obtaining and enriching or selecting a population of T cells from a biological sample for use as a first or input population of T cells. In some cases, the first population of T cells is one that is known or likely to contain T cells reactive to a tumor antigen or that are capable of being reactive to a tumor antigen, such as following an ex vivo co-culture with an autologous source of tumor antigen. For example, typically the first population of T cells is from a biological sample from a tumor or from a subject known or likely to have a tumor. In particular embodiments, the first population of T cells is further stimulated with one or more T cell stimulatory agent(s) (e.g. one or more recombinant cytokines, such as IL-2) and, in some cases one or more T cell adjuvant, to produce a second or stimulated population of T cells containing expanding T cells.

[0224] In some cases, conditions for stimulating the T cells by culture with one or more T cell stimulatory agent(s), and in some cases one or more T cell adjuvant, results in expansion or outgrowth of T cells present in the first or input population of T cells. In some embodiments, the conditions for stimulating the T cells with one or more T cell stimulatory agent(s), and in some cases one or more T cell adjuvant, can include culturing the T cells under condition that results in bulk expansion of the T cells. In other particular embodiments, conditions for stimulating the T cells may include culturing the T cells under conditions that are carried out to result in preferential or favored enrichment or outgrowth of desired T cells while minimizing or reducing certain T cell subsets that may not be desired.

[0225] In the provided methods, the stimulated composition of T cells is then employed in subsequent downstream steps for enrichment and expansion of tumor reactive T cells, including steps that include co-culture of the stimulated T cells with antigen presenting cells (APCs) in the presence of T cell neopitope (mutated) peptide antigens to produce, yield or to pull out T cells that are tumor reactive T cells. In particular embodiments, the provided methods also can include a step for selecting or enriching T cells reactive to a tumor antigen (tumor reactive T cells), after co-culturing T cells with APCs / peptide neoepitopes. The tumor reactive T cell populations can be cultured under conditions for expansion, such as to produce a therapeutic T cell composition.

[0226] In some embodiments, a biological sample is a sample from a subject having a tumor that is known to or is likely to contain tumor-reactive T cells for which such T cells have been exposed to or activated by a tumor neoantigen in vivo. In some embodiments, selecting the T cells from the biological sample further includes enriching or selecting for tumor-reactive T cells or T cells that express one or more activation markers associated with tumor-reactive T cells. The T cell activation markers include cell surface markers whose expression is upregulated or specific to T cells that have been exposed to antigen and activated. Exemplary markers are described in Section I.D below.

[0227] In aspects of any of the provided methods, the input or first population of T cells is incubated in the presence of a T cell stimulatory agent(s). In particular embodiments, the incubation is carried out under conditions in which the T cell stimulatory agent(s) activates or stimulates the cells or promotes expansion of T cells present in the input or first population of T cells.

[0228] In some embodiments, the T cell stimulatory agent(s) include 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 includes 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 cytokines are IL-7 and IL-15.

[0229] In some embodiments, the T cell stimulatory (agent(s) can include an agent or agents that engage CD3 and a costimulatory molecule, such as CD28. The T cell stimulatory agent(s) can include an anti-CD3 antibody, such as OKT3, and an anti-CD28 agent (presented by APC's or as a soluble antibody. In embodiments, prior to and / or during at least a portion of the co-culturing of T cells with APCs, the T cells selected from the biological sample (i.e. input or first population) are incubated in the presence of a T cell stimulatory agent(s), such as an anti-CD3 (e.g. OKT3) / anti-CD28 antibody. Thus, either before the co-culture in the presence of APCs or after the selection of reactive cells, the T cells are incubated with one or more T-cell stimulating agents of lymphocytes, such as but not limited to anti-CD3 antibody (e.g. OKT3) and anti-CD28 (presented by APC's or as soluble antibodies), to produce a second population of T cells that include activated or stimulated T cells. In particular embodiments, one or more recombinant cytokines also are present as additional T cell stimulatory agents during the incubation.

[0230] In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out directly on an input or first population of T cells selected from a biological sample from a subject, wherein the population of T cells selected from the biological sample (e.g. autologous T cells from the subject) is incubated with the T cell stimulatory agent(s). In other embodiments, the input population of T cells includes T cells that are likely to be or are suspected to be tumor reactive T cells, in which such cells are first selected from a population of T cells selected from a biological sample from a subject by selecting for cells positive for a surface marker that is upregulated on activated T cells (e.g. 4-1BB or OX40). In such embodiments, the incubation with the T cell stimulatory agent(s) is carried out after the enriching for the population of T cell cells comprising tumor-reactive T cells. In the provided embodiments, the incubation with the T cell stimulatory agent(s) is carried out before the co-culturing of such T cells (stimulated T cells) with the APCs / peptide neoepitopes.1. Selecting a Population of T cells

[0231] The provided methods include selecting or obtaining a population of T cells from a biological sample, which can be used as the source or input of T cells for stimulation with one or more T cell stimulatory agents(s) (e.g. recombinant IL-1 and / or anti-CD3 / anti-CD28) and, in provided embodiments, also a T cell modulatory agent (e.g. T cell agonist or apoptosis inhibitor). In some embodiments, the T cells are from a biological sample from a subject that is known or likely to contain tumor reactive T cells. The collected biological sample contains or is suspected to contain lymphocytes that have endogenous TCRs that are reactive to mutations present on a tumor.

[0232] In aspects of any of the provided embodiments, a suitable biological sample from a subject, such as from a patient of interest, i.e., a patient suspected of having or known to have cancer, is obtained. In some embodiments, the sample is one that is known or suspected of containing T cells, such as T cells that may be or may likely express an endogenous T cell receptor (TCR) specific to, binds or recognizes a tumor-associated antigen. The sample may be derived from any initial source that would contain or is suspected of containing such T cells. In some aspects, biological sample sources of interest include, but are not limited to, many different physiological sources, e.g. tissue derived samples, e.g. homogenates, and blood or derivatives thereof.

[0233] Any of a variety of samples can be used as a source of potentially reactive T cells. Although the tumor and downstream lymph nodes may have the highest frequency of reactive T cells (Powell et al., Clin. Cancer. Res., 2014), other sample sources also can be used. In some cases the sample is a tumor sample, a tertiary lymphoid 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.

[0234] The samples include tissue, fluid, and other samples taken directly from the 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. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0235] In some aspects, the sample is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0236] In many embodiments, the sample may be derived from fluids in which the T cells of interest are at least suspected of being present. 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 may be derived from whole blood or a fraction thereof, e.g. serum, plasma, etc., where in many embodiments the sample is derived from blood cells harvested from whole blood. In some aspects, the sample source contains mononuclear cells. For example, a biological sample is or contains peripheral blood mononuclear cells (PBMCs) or is derived from PBMCs.

[0237] In some embodiments in which the sample is a PBMC derived sample, the sample is generally a fluid PBMC derived sample. Any convenient methodology for producing a fluid PBMC sample may be employed. In many embodiments, the fluid PBMC derived sample is prepared by separating PBMCs from whole blood, i.e., collecting PBMCs, e.g., by centrifugation (such as by Ficoll-Hypaque density gradient centrifugation, where representative protocols for such separation procedures are disclosed in WO 98 / 15646 and U.S. Pat. No. 5,985,565).

[0238] In some embodiments, the sample is a tumor sample and thereby provides a source of tumor-infiltrating lymphocytes (TILs). In some aspects, TILs are T cells that have left the bloodstream of a subject and migrated into or infiltrated a tumor. In particular aspects, TILs are reactive to a tumor antigen.

[0239] A patient tumor sample may be obtained by any of a variety of methods in which the method obtains a sample that contains a mixture of tumor and TIL cells. In some embodiments, the tumor sample is obtained by surgical resection. In some embodiments, the tumor sample is obtained by needle biopsy. In general, the tumor sample may be from any solid tumor, including primary tumors, invasive tumors or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy. The solid tumor may be of any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, renal, stomach (gastrointestinal), and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In particular embodiments, the tumor is any as described in Section IV. In some embodiments, the tumor sample is from the same tumor source as was used to identify a neoantigen for preparing peptide neoepitopes.

[0240] In provided embodiments, the obtained tumor sample is fragmented into small pieces of between at or about 1 mm 3< and at or about 8 mm 3< in size, such as between at or about 1 mm 3< and at or about 6 mm 3< , between at or about 1 mm 3< and at or about 4 mm 3< , between at or about 1 mm 3< and at or about 2 mm 3< . In some embodiments, the tumor fragment is from about 2-3 mm 3< . In some embodiments, the tumor fragment is from about 1-2 mm 3< . In some embodiments, the tumor fragment is obtained by physical fragmentation, such as by dissection. In some embodiments, the tumor fragment is obtained by sharp dissection.

[0241] In some of any of the provided embodiments, the obtained tumor sample is fragmented into small pieces of between at or about 1 mm and at or about 8 mm in diameter, such as between at or about 1 mm and at or about 6 mm in diameter, between at or about 1 mm and at or about 4 mm in diameter, between at or about 1 mm and at or about 2 mm in diameter. In some embodiments, the tumor fragment is from about 2-3 mm in diameter. In some embodiments, the tumor fragment is from about 1-2 mm in diameter. In some embodiments, the tumor fragment is obtained by physical fragmentation, such as by dissection. In some embodiments, the tumor fragment is obtained by sharp dissection.

[0242] In some embodiments, the tumor sample is cryopreserved prior to fragmentation. In some embodiments, the tumor fragments are cryopreserved.

[0243] In some embodiments, obtained tumor fragments are placed into culture media under conditions and with appropriate nutrients to sustain T cell expansion, such as any of the conditions described in Subsection I.B.2 below for stimulation of T cells. In some embodiments 1 to 500 tumor fragments (e.g. each 1-8 mm in size) are placed in an appropriate culture vessel under conditions for expansion. In some embodiments, 10, 20, 30, 40, 50 or more fragments are cultured under conditions for expansion. The culture vessel can be a microwell, flask, tube, bag or other closed system device. In some embodiments the culture vessel is a closed container that provides a gas-permeable surface area, such as a a gas permeable flask. An exemplary culture vessel that provides a gas-permeable surface area include G-Rex plates or flasks. In some embodiments, 1 tumor fragment (about 1-8 mm in diameter) is placed for each about 2 cm 2< area of a culture vessel. The particular culture vessel can be chosen based on the number of tumor fragments available and / or the desired yield of cells. The choice of culture vessel (e.g. G-Rex) can be chosen by linearly scaling the number of fragments seeded to the surface area of the culture vessel. In some embodiments, the surface areas of the culture vessel is about 2 cm 2< (e.g. G-Rex 24 well plate) and about 1 tumor fragment (about 1-8 mm in diameter) is placed in the culture vessel. In some embodiments, the surface area of a 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 a 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 a culture vessel is about 500 cm 2< (e.g. G-Rex 500 M / 500M-CS) and about 250 tumor fragments (each about 1-8 mm in diameter) are placed in the culture vessel. In aspects of the provided methods, increasing the size of the culture vessel, and hence the number of tumor fragments per vessel, may decrease variability as compared to methods involving smaller culture vessels and / or fewer fragments per vessel, such by pooling larger numbers of fragments to minimize inter-tumor variability among fragments.

[0244] In some embodiments, the tumor fragments are placed in culture media for stimulation of the cells using any of the conditions described in Subsection I.B.2 below. In some embodiments, the culture media is a serum-free media containing recombinant cytokine from IL-2, IL-7, IL-15, and / or IL-21, such as recombinant IL-12 or recombinant IL-7 and IL-15. The concentration of recombinant cytokine can include any as described. In particular embodiments, the culture media is a serum-free media containing recombinant IL-2, such as from at or about 300 IU / mL to at or about 1000 IU / mL, for example at or about 300 IU / mL. In some embodiments, the culture media is a serum-free media containing an anti-CD3 antibody and / or CD28 targeting agent (e.g. anti-CD28 antibody) and one or more recombinant cytokines (e.g. IL-2). In some embodiments, the culture media contains one or more additional T cell stimulatory agonist or apoptosis inhibitor as described in Section II.

[0245] In some embodiments, the provided methods involve obtaining cells from the tumor fragments, such as by enzymatic digestion of tumor fragments to obtain TILs. Enzymatic digestion can be carried out using a collagenase, such as a type IV collagenase or a type I / II collagenase. The enzyme, such as a collagenase, can be present in media for the enzymatic digestion at a concentration of from at or about 1 mg / mL to at or about 5 mg / mL, such as at or about 1 mg / mL, at or about 2 mg / mL, at or about 3 mg / mL, at or about 4 mg / mL or at or about 5 mg / mL, or any value between any of the foregoing. In some embodiments, the enzymatic digestion is with a media that includes type IV collagenase, such as from at or about 1 mg / mL to at or about 5 mg / mL. In some embodiments, the enzymatic digestion is with a media that includes type I / II collagenase, such as from at or about 1 mg / mL to at or about 5 mg / mL. In other embodiments, enzymes from the Miltenyi human tumor dissociation kit can be used (e.g. Cat. O. 130-095-929; Miltenyi Biotec). The enzymatic media containing the enzyme can be a serum-free media, such as any as described. In particular embodiments, enzymatic media includes collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g. GlutaMAX), 10 mg / mL gentamicin, 30 units / mL of DNase and 1.0 mg / mL of collagenase). In some embodiments, enzymatic media includes a serum free media (e.g. OpTmizer) containing 2 mM glutamate (e.g. GlutaMAX), 10 µg / mL gentamicin, an immune cell serum replacement (e.g. CTS Immune Cell Serum Replacement) and 1.0 mg / mL to 5.0 mg / mL of collagenase). In some embodiments, the collagenase is a type IV collagenase. In some embodiments, the collagenase is a type I / II collagenase.

[0246] The tumor fragment is then mechanically dissected to dissociate the TILs, e.g., using a tissue dissociator. Tumor digests may be produced by placing the tumor in enzymatic media and mechanically dissociating the tumor for approximately 1 minute, followed by incubation for 30 minutes at 37 °C in 5% C0 2 , followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL can be performed to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 A1. Any of the foregoing methods may be used in any of the embodiments described herein for methods of obtaining TILs for use in the provided methods.

[0247] In some embodiments, digested cells from the tumor fragments are placed into culture media under conditions and with appropriate nutrients to sustain T cell expansion, such as any of the conditions described in Subsection I.B.2 below for stimulation of T cells. The cells are seeded at a particular density suitable 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 closed container that provides a gas-permeable surface area, such as a a gas permeable flask. An exemplary culture vessel that provides a gas-permeable surface area include G-Rex plates or flasks. In some embodiments approximately 5 x 10 5< to 2 x 10 6< cells of an enzymatically digested single cell suspension are seeded for each about 2 cm 2< area of a culture vessel. The particular culture vessel can be chosen based on the number of cells available and / or the desired yield of cells. The choice of culture vessel (e.g. G-Rex) can be chosen by linearly scaling the number of cells seeded to the surface area of the culture vessel. In some embodiments, the surface areas of the culture vessel is about 2 cm 2< (e.g. G-Rex 24 well plate) and about 5 x 10 5< to 2 x 10 6< cells of an enzymatically digested single cell suspension is placed in the culture vessel. In some embodiments, the surface area of a culture vessel is about 10 cm 2< (e.g. G-Rex 10 or G-Rex 10M) and about 2.5 x 10 6< to 1 x 10 7< cells of an enzymatically digested single cell suspension are placed in the culture vessel. In some embodiments, the surface area of a culture vessel is about 100 cm 2< (e.g. G-Rex 100 M / 100M-CS) and about 2.5 x 10 7< to 1 x 10 8< cells of an enzymatically digested single cell suspension are placed in the culture vessel. In some embodiments, the surface area of a culture vessel is about 500 cm 2< (e.g. G-Rex 500 M / 500M-CS) and about 1.25 x 10 8< to 5 x 10 8< cells of an enzymatically digested single cell suspension are placed in the culture vessel.

[0248] In some embodiments, the culture media is a serum-free media containing recombinant IL-2. In some embodiments, one or more additional T cell stimulating agent can also be included. In some embodiments, the culture media is a serum-free media containing an anti-CD3 antibody and / or a CD28 targeting agent (e.g. anti-CD28 antibody) and one or more recombinant cytokines (e.g. IL-2). In some embodiments, the culture media contains one or more additional T cell stimulatory agonist or apoptosis inhibitor as described in Section II.

[0249] The sample may be obtained from a variety of different subjects / patients / hosts. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (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 hosts will be humans.

[0250] In some aspects, the subject is a human. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. In some embodiments, the sample is autologous to a subject to be treated, such as a subject who is a patient in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or expanded in accord with the provided methods. In some embodiments, the sample is allogenic to a subject to be treated.

[0251] In some embodiments, the T cells for use in connection with the provided methods can be enriched or sorted a variety of ways including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed cartridge based cell sorters. In particular aspects, one or more reagents specific to T cells or a subset thereof, such as reagents specific to T cell activation markers for selecting reactive cells, can be used including, but not limited to, florescent antibodies, nanoparticles or beads on cell selection equipment, but not limited to, the CliniMACS, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0252] In some aspects, T cells can be selected from a biological sample, such as based on T cell markers CD3, CD4 or CD8. In some embodiments, selecting for a T cell that is surface positive for one or more cell surface marker includes any method for separation based on such markers.

[0253] In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. In some embodiments, the immunoaffinity-based selections include contacting a sample containing cells, such as a sample containing a bulk population of T cells, e.g. primary human T cells, containing CD3+ T cells or CD4+ and CD8+ cells, with an antibody or binding partner that specifically binds to the cell surface marker or markers. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a sphere or bead, for example a nanoparticle, microbeads, nanobeads, including agarose, magnetic bead or paramagnetic beads, to allow for separation of cells for positive and / or negative selection. In some embodiments, the spheres or beads can be packed into a column to effect immunoaffinity chromatography, in which a sample containing cells, such as primary human T cells containing CD3+ T cells or 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.

[0254] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as nanoparticles or paramagnetic beads. The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select. Such beads are known and are commercially available from a variety of sources including, in some aspects, 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 those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained.

[0255] In certain embodiments, the sample is contacted with a binding agent, e.g., a detectably labeled binding agent, that specifically binds to a cell surface marker. In certain embodiments, the detectably labeled binding agent(s) are fluorescently labeled. In certain embodiments, T cells labeled with binding agents specific to a cell surface marker are identified by flow cytometry. In certain embodiments, the method further includes separating any resultant T cells labeled with the binding agent(s) from other components of the sample to produce a composition enriched for T cells surface positive for the one or more cell surface marker. Cell selection sorting equipment can be used that has a sufficiently high-throughput to handle large volumes and cell numbers. Non-limiting cell sorting equipment includes, for example, Sony FX500 or the Tyto cell sorting systems (Miltenyi).

[0256] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead and / or are detectably labeled, specifically bind to cell surface molecules if present on cells within the sample. In some aspects, cells bound to the antibodies can be recovered or separated from non-bound cells in the sample.

[0257] 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 subject to further separation steps. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0258] The separation need not result in 100 % enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells. For example, in some aspects, a selection of one of the CD4+ or CD8+ population enriches for said population, either the CD4+ or CD8+ population, but also can contain some residual or small percentage of other non-selected cells, which can, in some cases, include the other of the CD4 or CD8 population still being present in the enriched population.

[0259] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker +< ) at a relatively higher level (marker high< ) on the positively or negatively selected cells, respectively.

[0260] In particular embodiments, a T cell population is one that includes both CD4+ and CD8+ T cells. In some cases, a CD4+ and CD8+ T cell population is isolated, selected or enriched from the biological sample. Many cancers, including solid tumors, such as many common epithelial indications (e.g. GI), express class I and class II restricted mutations. In order for a T cell product to target such indications, e.g. common epithelial indications, it is contemplated that both CD8+ T cells to recognize class I MHC-restricted molecules and CD4+ T cells to recognize Class II MHC-restricted molecules are necessary.

[0261] In some embodiments, the methods include isolation, selection and / or enrichment of CD3+ cells. In some embodiments, the methods include isolation, selection and / or enrichment of CD4+ and CD8+ cells. In some aspects, a CD4 +< or CD8 +< selection step, such as positive selection for CD4 and positive selection for CD8, is used to separate CD4 +< helper and CD8 +< cytotoxic T cells. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the methods include enriching for CD4+ and CD8+ T cells by selecting for T cells surface positive for CD3 or by sequential or simultaneous selection for T cells surface positive for CD4 and T cells surface positive for CD8. Such CD3+ T cells, or CD4 -< and / or CD8 +< populations, can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on tumor-reactive T cells or on T cells having expression of T cell activation markers associated with tumor-reactive T cells, e.g. as described in Section I.D..

[0262] In some embodiments, isolation of the cells or populations further includes one or more preparation and / or non-affinity based cell separation steps. In some examples, 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, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0263] 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 greater than about 60%, greater than or greater than about 70%, greater than or greater than about 80%, greater than or greater than about 90% or greater than or greater than about 95%. In some embodiments, the selected population is enriched for CD4+ T cell 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 greater than about 60%, greater than or greater than about 70%, greater than or greater than about 80%, greater than or greater than about 90% or greater than or greater than about 95%. In particular embodiments, the ratio of CD8+ T cells to CD4+ T cells is between at or about 1:100 and at or about 100:1, between at or about 1:50 and at or about 50:1, between at or about 1:25 and at or about 25: 1, between at or about 1:10 and at or about 10: 1, between at or about 1:5 and at or about 5:1, or between at or about 1:2.5 and at or about 2.5:1.

[0264] In some of any of the provided embodiments, the biological sample is a peripheral blood sample, optionally an apheresis sample, and wherein: the number of cells at the initiation of the culturing is between at or about 1 x 10 9< and 7 x 10 9< total viable cells; or is at or about 1 x 10 9< total viable cells, at or about 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< 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 culturing is between at or about 0.02% and at or about 40%, at or about 0.02% and at or about 24%, at or about 0.02% and at or about 18%, at or about 0.02% and at or about 0.9% or at or about 0.02% and at or about 6.0%; and / or the number of T cells surface positive for the T cell activation marker at the initiation of the culturing is between at or about 0.1 x 10 6< and at or about 60 x 10 6< T cells, 0.1 x 10 6< and at or about 8 x 10 6< T cells, 0.1 x 10 6< and at or about 20 x 10 6< T cells, 0.3 x 10 6< and at or about 35 x 10 6< T cells or 0.3 x 10 6< and at or about 60 x 10 6< T cells; or is at or about 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 foregoing.

[0265] In some of any of the provided embodiments, the biological sample is a lymph sourced sample or a tumor sourced sample, and wherein: the number of cells at the initiation of the culturing is between at or about 10 x 10 6< and 100 x 10 6< total viable cells, 20 x 10 6< and 100 x 10 6< total viable cells, or 12 x 10 6< and 43 x 10 6< total viable cells; or is at or about 10 x 10 6< total viable cells, at or about 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 culturing is between at or about 1% and at or about 90%, at or about 1% and at or about 75%, at or about 1% and at or about 50%, at or about 1% and at or about 25% or at or about 1% and at or about 14%; and / or the number of T cells surface positive for the T cell activation marker at the initiation of the culturing is between at or about 0.7 x 10 6< and at or about 15 x 10 6< T cells, 1 x 10 6< and at or about 15 x 10 6< T cells, or at or about 0.7 x 10 6< and at or about 5.4 x 10 6< T cells; or is at or about 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 foregoing.

[0266] In some embodiments, the selected T cells can be further enriched for tumor-reactive T cells based on expression of a marker associated with activated T cells. Particular markers for use in selecting or enriching for such tumor-reactive T cells is described in Section I.D. below. In other cases, selection or enrichment of tumor-reactive T cells is carried out in one or more subsequent step of the process, such as after co-culture with one or more mutated peptide (peptide neoepitope).2. Stimulation of T cells for Initial Expansion

[0267] In aspects of the provided methods, the T cells from the biological sample (input or first population of T cells, such as present in a resected tumor fragment) are incubated or cultured in the presence of one or more T cell stimulatory agent(s) under conditions for stimulating the T cells, such as to expand the T cells. In some cases, the culturing or incubation is further carried out in the presence of one or more T cell modulatory agent or adjuvant, such as a T cell agonist or apoptosis inhibitor. In some embodiments, the incubation or culturing with one or more T cell stimulatory agent(s) and / or T cell modulatory agent or adjuvants results in expansion or outgrowth of selected T cells, or a desired subset or subtype thereof or for viable cells thereof, for use in subsequent steps of the provided methods. Non-limiting examples of T cell stimulatory agent(s) and / or T cell modulatory agents or adjuvants and conditions for incubation or culture are described herein.

[0268] In some embodiments, the T cell stimulatory agent(s) include 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 includes 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 includes IL-7 and IL-15.

[0269] In aspects of any of the provided methods, the population of T cells is incubated in the presence of a T cell stimulatory agent(s). In particular embodiments, the incubation is carried out under conditions in which the T cell stimulatory agent(s) activates or stimulates the cells or promotes expansion of cells.

[0270] In some of any of the provided embodiments, the T cell stimulatory agent(s) is selected from an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling via a costimulatory receptor. In some of any of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, such as OKT3. In some of any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor comprises peripheral blood mononuclear cells (PBMCs), optionally non-dividing or irradiated PBMCs. In some of any of the provided embodiments, the agent that initiates signaling via a costimulatory receptor is an anti-CD28 antibody. In some of any of the provided embodiments, the T cell stimulatory agent(s) is an anti-CD3 antibody and an anti-CD28 antibody that each are soluble.

[0271] Thus, among the provided methods are methods of culturing T cells for manufacture of tumor reactive T cells in which T cells are cultured or incubated in the presence of a T cell stimulatory agent under conditions to expand T cells, such as present in the co-culture. In some embodiments, the T cell stimulatory agent is or includes an anti-CD3 antibody and anti-CD28 antibody.

[0272] In embodiments of the provided methods, the stimulating conditions include one or more agent, e.g., ligand, which turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell and / or a costimulatory signal in a T cell. Such agents can include antibodies, such as those specific for a TCR component, e.g., anti-CD3, and / or costimulatory receptor, 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 solid support such as a bead. In some embodiments, the T cell stimulatory agent(s) includes anti-CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS ®< M-450 CD3 / CD28 T Cell Expander).

[0273] An anti-CD3 antibody can include any antibody directed against or that can specifically bind 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 theUHCTI clone, also known as T3 and CD3E. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. The anti-CD3 antibody can be added as a soluble reagent or bound to a bead. In particular embodiments, the anti-CD3 antibody is soluble.

[0274] In particular embodiments, the T cell stimulatory agent(s) include an anti-CD3 antibody, which is added to the cell culture medium during the incubation. In some embodiments, the anti-CD3 antibody is added at a concentration ranging between at or about 0.1 ng / mL and 50 ng / mL, such between at or about 0.5 ng / mL and at or about 50 ng / mL, between at or about 0.5 ng / mL and at or about 30 ng / mL, between at or about 0.5 ng / mL and at or about 15 ng / mL, between at or about 0.5 ng / mL and at or about 5 ng / mL, between at or about 0.5 ng / mL and at or about 1 ng / mL, between at or about 1 ng / mL and at or about 50 ng / mL, between at or about 1 ng / mL and at or about 30 ng / mL, between at or about 1 ng / mL and at or about 15 ng / mL, between at or about 1 ng / mL and at or about 5 ng / mL, between at or about 5 ng / mL and at or about 50 ng / mL, between at or about 5 ng / mL and at or about 30 ng / mL, between at or about 5 ng / mL and at or about 15 ng / mL, between at or about 15 ng / mL and at or 50 ng / mL, between at or about 15 ng / mL and at or about 30 ng / mL or between at or about 30 ng / mL and at or about 50 ng / mL, each inclusive.

[0275] In particular embodiments, the anti-CD3 antibody is OKT3. In an embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 µg / mL of OKT3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT3 antibody.

[0276] In some embodiments, the T cell stimulatory agent(s) includes incubation with an anti-CD3 antibody and incubation with a further agent that specifically binds to CD28 or stimulates or induces a CD28-mediated signal in cells. In some embodiments, the CD28-mediated signal can be initiated or provided by anti-CD28 antibody or antigen-binding fragment thereof. In some embodiments, the CD28-mediated signal can be provided by antigen-presenting feeder cells (APCs), such as peripheral blood mononuclear cells (PBMC).

[0277] In some embodiments, the T cell stimulatory agent(s) can include adding to the population of T cells feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC). In some aspects, the non-dividing feeder cells can comprise gamma- irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells. In some embodiments, the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded. In some embodiments, the ratio of T cells to PBMCs and / or antigen-presenting cells is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500.

[0278] In some embodiments, the T cell stimulatory agent(s) can include adding adding to the population of cells an anti-CD28 antibody or antigen-binding fragment thereof. An anti-CD28 antibody can include any antibody directed against or that can specifically bind the CD28 receptor on the surface of T cells. Non-limiting examples of anti-CD28 antibodies include NA / LE (e.g. BD Pharmingen), IM1376 (e.g. Beckman Coulter), or 15E8 (e.g. Miltenyi Biotec). The anti-CD28 antibody can be added as a soluble reagent or bound to a bead. In particular embodiments, the anti-CD3 antibody is soluble. In some embodiments, the anti-CD28 antibody is added at a concentration ranging between at or about 1 ng / mL and 1000 ng / mL, between at or about 1 ng / mL and 500 ng / mL, between at or about 1 ng / mL and at or about 100 ng / mL, between at or about 1 ng / mL and at or about 10 ng / mL, between at or about 10 ng / mL and at or about 1000 ng / mL, between at or about 10 ng / mL and at or about 500 ng / mL, between at or about 10 ng / mL and at or about 100 ng / mL, between at or about 100 ng / mL and at or about 1000 ng / mL, between at or about 100 ng / mL and at or about 500 ng / mL or between at or about 500 ng / mL and at or about 1000 ng / mL.

[0279] In some embodiments, the T cell stimulatory agent(s) include one or more recombinant cytokine. In some embodiments, the cytokine is added or is exogenous to the culture media. In some embodiments, the recombinant cytokine can include one or more of IL-2, IL-7, IL-15 or IL-21. In some embodiments, the culturing and incubation is carried out in the presence of recombinant IL-2, IL-15 and IL-7. In some embodiments, the culturing is carried out in the presence of a IL-2. In some embodiments, the culturing is carried out in the presence of IL-15 and IL-7, which, in some aspects does not additionally include IL-2. In particular embodiments, the recombinant cytokine(s) is human.

[0280] The recombinant cytokine generally is a recombinant human protein. In particular embodiments, the recombinant cytokine is present in the cell culture medium during the incubation at a concentration of at least at or about or at or about 10 IU / mL, at least at or about or at or about 100 IU / mL, at least at or about or at or about 1000 IU / mL, at least at or about or at or about 1500 IU / mL, at least at or about or at or about 2000 IU / mL, at least at or about or at or about 2500 IU / mL, at least at or about or at or about 3000 IU / mL, at least at or about or at or about 3500 IU / mL, at least at or about or at or about 4000 IU / mL, at least at or about or at or about 4500 IU / mL, at least at or about or at or about 5000 IU / mL, at least at or about or at or about 5500 IU / mL, at least at or about or at or about 6000 IU / mL, at least at or about or at or about 6500 IU / mL, at least at or about or at or about 7000 IU / mL, at least at or about or at or about 7500 IU / mL, or at least at or about or at or about 8000 IU / mL. In an embodiment, the cell culture medium comprises between at or about 10 IU / mL and at or about 100 IU / mL, at or about 100 IU / mL and at or about 1000 IU / mL, at or about 1000 and at or about 2000 IU / mL, between at or about 2000 and at or about 3000 IU / mL, between at or about 3000 and 4000 at or about IU / mL, between at or about 4000 and at or about 5000 IU / mL, between at or about 5000 and at or about 6000 IU / mL, between at or about 6000 and at or about 7000 IU / mL, between at or about 7000 and at or about 8000 IU / mL, each inclusive.

[0281] In some embodiments, recombinant IL-2 is present in the cell culture medium. IL-2 is a cytokine that supports T cell recovery and proliferation. IL-2 also supports the homeostasis of T cells, thereby supporting their phenotype, differentiation status, and immune memory. In some cases, induction of regulatory T cells in the tumor microenvironment may lead to low bioavailability of IL-2. Recombinant IL-2 has been regularly used in broad expansion of T cells in various contexts. Recombinant IL-2 is commercially available. In particular embodiments, recombinant IL-2 is GMP grade (e.g. MACS GMP Recombinant Human IL-2, Miltenyi Biotec).

[0282] Recombinant IL-2 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-2 can be included in the initial T cell expansion (first expansion), such as to promote TIL outgrowth and allow their proliferation from solid tumor. IL-2 also can be included in antigen-presenting cell co-culture as described in Section I.C, such as to allow for peak activation of neo-antigen reactive T prior to their separation or selection. In some cases, recombinant IL-2 can also be included in cultures to expand tumor-reactive T cells during the second expansion phase, such as described in Section I.E.

[0283] In some embodiments, recombinant IL-2 is added to the culture medium at a concentration between at or about 10 IU / mL and at or about 1000 IU / mL, such as between at or about 10 IU / mL and at or about 600 IU / mL, between at or about 10 IU / mL and at or about 400 IU / mL, between at or about 10 IU / mL and at or about 200 IU / mL, between at or about 10 IU / mL and at or about 100 IU / mL, between at or about 10 IU / mL and at or about 50 IU / mL, between at or about 50 IU / mL and at or about 1000 IU / mL, between at or about 50 IU / mL and at or about 600 IU / mL, between at or about 50 IU / mL and at or about 400 IU / mL, between at or about 50 IU / mL and at or about 200 IU / mL, between at or about 50 IU / mL and at or about 100 IU / mL, between at or about 100 IU / mL and at or about 1000 IU / mL, between at or about 100 IU / mL and at or about 600 IU / mL, between at or about 100 IU / mL and at or about 400 IU / mL, between at or about 100 IU / mL and at or about 200 IU / mL, between at or about 200 IU / mL and at or about 1000 IU / mL, between at or about 200 IU / mL and at or about 600 IU / mL, between at or about 200 IU / mL and at or about 400 IU / mL, between at or about 400 IU / mL and at or about 1000 IU / mL, between at or about 400 IU / mL and at or about 600 IU / mL or between at or about 600 IU / mL and at or about 1000 IU / mL. In some embodiments, recombinant IL-2 is present in an amount that is between 50 and 400 IU / mL.

[0284] In some embodiments, the incubation is carried out with a higher dose IL-2. In some aspects, IL-2 is the only recombinant cytokine added to the culture. In some embodiments, the recombinant IL-2 is added to the culture medium at a concentration between at or about 1000 IU / mL at at or about 8000 IU / mL, such as between at or about 1000 IU / mL and at or about 7000 IU / mL, between at or about 1000 IU / mL and at or about 6000 IU / mL, between at or about 1000 IU / mL and at or about 5000 IU / mL, between at or about 1000 IU / mL and at or about 4000 IU / mL, between at or about 1000 IU / mL and at or about 2000 IU / mL, 2000 IU / mL at at or about 8000 IU / mL, between at or about 2000 IU / mL and at or about 7000 IU / mL, between at or about 2000 IU / mL and at or about 6000 IU / mL, between at or about 2000 IU / mL and at or about 5000 IU / mL, between at or about 2000 IU / mL and at or about 4000 IU / mL, 4000 IU / mL at at or about 8000 IU / mL, between at or about 4000 IU / mL and at or about 7000 IU / mL, between at or about 4000 IU / mL and at or about 6000 IU / mL, between at or about 4000 IU / mL and at or about 5000 IU / mL, between at or about 5000 IU / mL at at or about 8000 IU / mL, between at or about 5000 IU / mL and at or about 7000 IU / mL, between at or about 5000 IU / mL and at or about 6000 IU / mL, between at or about 6000 IU / mL at at or about 8000 IU / mL, between at or about 6000 IU / mL and at or about 7000 IU / mL or between at or about 7000 IU / mL and at or about 8000 IU / mL. In some embodiments, recombinant IL-2 is present in an amount that is or is about 6000 IU / mL.

[0285] In some embodiments, recombinant IL-15 is present in the cell culture medium. IL-15 is a cytokine that is involved in memory T cell homeostasis and activation. In some cases, IL-15 can promote effector functions of antigen-experienced T cells in the absence of antigen and prevent their differentiation into an exhausted phenotype. IL-15 also plays a role in T cell proliferation. Recombinant IL-15 is commercially available. In particular embodiments, recombinant IL-15 is GMP grade (e.g. MACS GMP Recombinant Human IL-15, Miltenyi Biotec).

[0286] Recombinant IL-15 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-15 can be included in the initial T cell expansion (first expansion), such as to promote TIL expansion to promote their outgrowth and allow their proliferation and / or stabilize phenotype from solid tumor. Recombinant IL-15 also can be included in antigen-presenting cell co-culture as described in Section I.C, such as to allow for peak activation of neo-antigen reactive T prior to their separation or selection. In some cases, recombinant IL-15 can also be included in cultures to expand tumor-reactive T cells during the second expansion phase, such as described in Section I.E. In some cases, recombinant IL-15 can be combined with recombinant IL-7 to provide for activation, survival and / or expansion of tumor-reactive T cells in the provided methods. In some such embodiments, the combination of recombinant IL-7 and IL-15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0287] In some embodiments, the recombinant IL-15 is added to the culture medium at a concentration between at or about 10 IU / mL and 500 IU / mL, such as between at or about 10 IU / mL and at or about 400 IU / mL, between at or about 10 IU / mL and at or about 300 IU / mL, between at or about 10 IU / mL and at or about 200 IU / mL, between at or about 10 IU / mL and at or about 100 IU / mL, between at or about 10 IU / mL and at or about 70 IU / mL, between at or about 10 IU / mL and at or about 50 IU / mL, between at or about 10 IU / mL and at or about 30 IU / mL, between at or about 30 IU / mL and 500 IU / mL, between at or about 30 IU / mL and at or about 400 IU / mL, between at or about 30 IU / mL and at or about 300 IU / mL, between at or about 30 IU / mL and at or about 200 IU / mL, between at or about 30 IU / mL and at or about 100 IU / mL, between at or about 30 IU / mL and at or about 70 IU / mL, between at or about 30 IU / mL and at or about 50 IU / mL, between at or about 50 IU / mL and at or about 400 IU / mL, between at or about 50 IU / mL and at or about 500 IU / mL, between at or about 50 IU / mL and at or about 300 IU / mL, between at or about 50 IU / mL and at or about 200 IU / mL, between at or about 50 IU / mL and at or about 100 IU / mL, between at or about 50 IU / mL and at or about 70 IU / mL, between at or about 70 IU / mL and at or about 500 IU / mL, between at or about 70 IU / mL and at or about 400 IU / mL, between at or about 70 IU / mL and at or about 300 IU / mL, between at or about 70 IU / mL and at or about 200 IU / mL, between at or about 70 IU / mL and at or about 100 IU / mL, between at or about 100 IU / mL and at or about 500 IU / mL, between at or about 100 IU / mL and at or about 400 IU / mL, between at or about 100 IU / mL and at or about 300 IU / mL, between at or about 100 IU / mL and at or about 200 IU / mL, between at or about 200 IU / mL and at or about 500 IU / mL, between at or about 200 IU / mL and at or about 400 IU / mL, between at or about 200 IU / mL and at or about 300 IU / mL, between at or about 300 IU / mL and at at or about 500 IU / mL, between at or about 200 IU / mL and at or about 400 IU / mL, or between at or about 400 IU / mL and at or about 500 IU / mL. In some embodiments, the IL-15 is added to the culture medium in an amount between at or about 100 IU / mL and at or about 200 IU / mL. In some embodiments, the IL-15 is added to the culture medium at or about 180 IU / mL.

[0288] In some embodiments, recombinant IL-7 is added to the culture medium. IL-7 is a cytokine that is involved in promoting T cell maintenance and homeostasis. In some cases, IL-7 can boost memory T cell survival and proliferation, particularly the central memory compartment. Recombinant IL-7 is commercially available. In particular embodiments, recombinant IL-7 is GMP grade (e.g. MACS GMP Recombinant Human IL-7, Miltenyi Biotec).

[0289] Recombinant IL-7 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-7 can be included in the initial T cell expansion (first expansion), such as to promote TIL expansion to promote their outgrowth and allow their proliferation and / or stabilize phenotype from solid tumor. IL-7 also can be included in antigen-presenting cell co-culture as described in Section I.C, such as to allow for peak activation of neo-antigen reactive T prior to their separation or selection. In some cases, recombinant IL-7 can also be included in cultures to expand tumor-reactive T cells during the second expansion phase, such as described in Section I.E. Inclusion of recombinant IL-7 in the process can maintain or support expansion of memory T cell subsets in the process. In some cases, recombinant IL-7 can be combined with recombinant IL-15 to provide for activation, survival and / or expansion of tumor-reactive T cells in the provided methods. In some such embodiments, the combination of recombinant IL-7 and IL-15 is an alternative to the use of recombinant IL-2 in the culture, and the culture media does not additionally contain recombinant IL-2.

[0290] In some embodiments, the recombinant IL-7 is added to the culture medium at a concentration between at or about 100 IU / mL and at or about 2000 IU / mL, between at or about 100 IU / mL and at or about 1500 IU / mL, between at or about 100 IU / mL and at or about1000 IU / mL, between at or about 100 IU / mL and at or about 800 IU / mL, between at or about 100 IU / mL and at or about 600 IU / mL, between at or about 100 IU / mL and at or about 400 IU / mL, between at or about 100 IU / mL and at or about 200 IU / mL, between at or about 200 IU / mL and at or about 2000 IU / mL, between at or about 200 IU / mL and at or about 1500 IU / mL, between at or about 200 IU / mL and at or about1000 IU / mL, between at or about 200 IU / mL and at or about 800 IU / mL, between at or about 200 IU / mL and at or about 600 IU / mL, between at or about 200 IU / mL and at or about 400 IU / mL, between at or about 400 IU / mL and at or about 2000 IU / mL, between at or about 400 IU / mL and at or about 1500 IU / mL, between at or about 400 IU / mL and at or about1000 IU / mL, between at or about 400 IU / mL and at or about 800 IU / mL, between at or about 400 IU / mL and at or about 600 IU / mL, between at or about 600 IU / mL and at or about 2000 IU / mL, between at or about 600 IU / mL and at or about 1500 IU / mL, between at or about 600 IU / mL and at or about1000 IU / mL, between at or about 600 IU / mL and at or about 800 IU / mL, between at or about 800 IU / mL and at or about 2000 IU / mL, between at or about 800 IU / mL and at or about 1500 IU / mL, between at or about 800 IU / mL and at or about 1000 IU / mL, between at or about 1000 IU / mL and at or about 2000 IU / mL, between at or about 1000 IU / mL and at or about 1500 IU / mL, between at or about 1500 IU / mL and at or about 2000 IU / mL. In some embodiments, the IL-7 is added to the culture medium in an amount between at or about 1000 IU / mL and at or about 2000 IU / mL. In some embodiments, the IL-7 is added to the culture medium at or about 600 IU / mL.

[0291] In some embodiments, recombinant IL-21 is added to the culture medium. IL-21 is a cytokine that supports a broad range of T cell activation without increasing regulatory T cell signaling. In some cases, IL-21 can support memory cell stabilization, effector function, and proliferation of antigen-experienced T cells. IL-21 can induce upregulation of effector molecules in both CD4 and CD8 T cells. Recombinant IL-21 is commercially available. In particular embodiments, recombinant IL-21 is GMP grade (e.g. MACS GMP Recombinant Human IL-21, Miltenyi Biotec).

[0292] Recombinant IL-21 can be included in cell culture media during various stages of the provided process. In some cases, recombinant IL-21 can be included in the initial T cell expansion (first expansion), such as to promote TIL outgrowth from solid tumor, including by stabilizing memory T cell activation, function and / or proliferation. In some aspects, the presence of IL-21 allows for improved recovery of TIL. Recombinant IL-21 also can be included in antigen-presenting cell co-culture as described in Section I.C, such as due to the ability to stimulate expression of T cell activation markers, including expression of activation markers on neo-antigen reactive TIL. In some cases, recombinant IL-21 can also be included in cultures to expand tumor-reactive T cells during the second expansion phase as described in Section I.E, such as to support proliferation and stabilization of memory phenotype.

[0293] In some embodiments, the recombinant IL-21 is added to the culture medium at a concentration between at or about 0.5 IU / mL and at or about 20 IU / mL, between at or about 0.5 IU / mL and at or about 15 IU / mL, between at or about 0.5 IU / mL and at or about 10 IU / mL, between at or about 0.5 IU / mL and at or about 5 IU / mL, between at or about 0.5 IU / mL and at or about 2.5 IU / mL, between at or about 0.5 IU / mL and at or about 1 IU / mL, between at or about 1 IU / mL and at or about 20 IU / mL, between at or about 1 IU / mL and at or about 15 IU / mL, between at or about 1 IU / mL and at or about 10 IU / mL, between at or about 1 IU / mL and at or about 5 IU / mL, between at or about 1 IU / mL and at or about 2.5 IU / mL, between at or about 2.5 IU / mL and at or about 20 IU / mL, between at or about 2.5 IU / mL and at or about 15 IU / mL, between at or about 2.5 IU / mL and at or about 10 IU / mL, between at or about 2.5 IU / mL and at or about 5 IU / mL, between at or about 5 IU / mL and at or about 20 IU / mL, between at or about 5 IU / mL and at or about 15 IU / mL, between at or about 5 IU / mL and at or about 10 IU / mL, between at or about 10 IU / mL and at or about 20 IU / mL, between at or about 10 IU / mL and at or about 15 IU / mL, or between at or about 15 IU / mL and at or about 20 IU / mL. In some embodiments, the IL-21 is added to the culture medium in an amount between at or about 0.5 IU / mL and at or about 2.5 IU / mL. In some embodiments, the IL-21 is added to the culture medium at or about 1 IU / mL.

[0294] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells, contains recombinant IL-2. In some embodiments, one or more other stimulating agent can be included such as one or more other recombinant cytokine from IL-7, IL-15, IL-21 or an anti-CD3 antibody (e.g. OKT-3). In some cases in which an anti-CD3 antibody (e.g. OKT-3) the T cell stimulating agent(s) also can include a costimulating agent, such as provided by antigen-presenting feeder cells, such as PBMCs, or a soluble anti-CD28 antibody.

[0295] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0296] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2, an anti-CD3 antibody, e.g. OKT-3, and an anti-CD28 antibody. In some embodiments, the anti-CD3 antibody and / or anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and anti-CD28 antibody are bound to a solid surface, such as a bead (e.g., DYNABEADS ®< M-450 CD3 / CD28 T Cell Expander).

[0297] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2, recombinant IL-15, recombinant IL-7, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0298] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-2, recombinant IL-15, recombinant IL-7, an anti-CD3 antibody, e.g. OKT-3, and an anti-CD28 antibody. In some embodiments, the anti-CD3 antibody and / or anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and anti-CD28 antibody are bound to a solid surface, such as a bead (e.g., DYNABEADS ®< M-450 CD3 / CD28 T Cell Expander).

[0299] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-15 and recombinant IL-7, an anti-CD3 antibody, e.g. OKT-3, and antigen-presenting feeder cells, such as PBMCs.

[0300] In particular embodiments, T cell stimulatory agent(s) present during the incubation, such as for expansion of cells contains recombinant IL-15 and recombinant IL-7, an anti-CD3 antibody, e.g. OKT-3, and an anti-CD28 antibody. In some embodiments, the anti-CD3 antibody and / or anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and anti-CD28 antibody are bound to a solid surface, such as a bead (e.g., DYNABEADS ®< M-450 CD3 / CD28 T Cell Expander).

[0301] In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out under conditions for initial expansion of T cells from the biological sample. In some embodiments, the cells are cultured at about 37 °C with about 5% CO 2 .

[0302] In some embodiments, the incubation with the T cell stimulatory agent(s) is carried out for at or about 1 day, such as generally at or about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or any range of time between any of the foregoing. In some embodiments, the incubation is carried out for 7-10 days. In some embodiments, the incubation is for at or about 7 days. In some embodiments, the incubation is for at or about 8 days. In some embodiments, the incubation is for at or about 9 days. In some embodiments, the incubation is for at or about 10 days.

[0303] The incubation, such as for initial expansion of T cells in the biological sample, can be carried out under GMP conditions. In some embodiments, the incubation is in a closed system, which in some aspects may be a closed automated system. In some embodiments, the culture media containing the T cell stimulatory agent(s) can be a serum-free media. In some embodiments, the incubation is carried out in a closed automated system and with serum-free media.

[0304] In some embodiments, the initial expansion of cells under the one or more stimulatory conditions is in a culture vessel suitable for cell expansion. In some embodiments, the culture vessel is a gas permeable culture vessel, such as a G-Rex system (e.g. G-Rex 10, G-Rex 10M, G-Rex 100 M / 100M-CS or G-Rex 500 M / 500M-CS). In some embodiments the culture vessel is a microplate, flask, bar or other culture vessel suitable for expansion of cells in a closed system. In some embodiments, expansion can be carried out in a bioreactor. In some embodiments, the initial expansion can be carried out using a cell expansion system by transfer of the cells to gas permeable bags, such as in connection with a bioreactor (e.g. Xuri Cell Expansion System W25 (GE Healthcare)). In an embodiment, the cell expansion system includes a culture vessel, such as a bag, e.g. gas permeable cell bag, with a volume that is about 50 mL, about 100 mL, about 200 mL, about 300 mL, about 400 mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, about 1 L, about 2 L, about 3 L, about 4 L, about 5 L, about 6 L, about 7 L, about 8 L, about 9 L, and about 10 L, or any value between any of the foregoing. In some embodiments, the process is automated or semi-automated. Examples of suitable bioreactors for the automated perfusion expansion include, but are not limited to, GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM 20 | 50, Finesse SmartRocker Bioreactor Systems, and Pall XRS Bioreactor Systems, or Miltenyi Prodigy. In some aspects, the expansion culture is carried out under static conditions. In some embodiments, the expansion culture is carried out under rocking conditions. The medium can be added in bolus or can be added on a perfusion schedule. In some embodiments, the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L / min, 0.05 L / min, 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 1.0 L / min, 1.5 L / min, or 2.0 L / min or greater than 2.0 L / min. In certain embodiments, at least a portion of the culturing is performed with perfusion, such as with a rate of 290 ml / day, 580 ml / day, and / or 1160 ml / day.

[0305] In some embodiments, the cells are seeded in an appropriate culture vessel (e.g. gas permeable bag) at a density of from 0.5 x 10 6< cells / mL to 1.5 x 10 6< cells / mL. In some embodiments, the density is at or about 0.5 x 10 6< cells / mL, 0.75 x 10 6< cells / mL, 1 x 10 6< cells / mL, 1.25 x 10 6< cells / mL or 1.5 x 10 6< cells / mL, or any value between any of the foregoing.

[0306] In some aspects, cells are expanded in an automated closed expansion system that is perfusion enabled. Perfusions can continuously add media to the cells to ensure an optimal growth rate is achieved.

[0307] The expansion methods can be carried out under GMP conditions, including in a closed automated system and using serum free medium. In some embodiments, any one or more of the steps of the method can be carried out 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 for carrying out one or more of the other processing steps of a method for manufacturing, generating or producing a cell therapy. In some embodiments, one or more or all of the processing steps, e.g., isolation, selection and / or enrichment, processing, culturing steps including incubation in connection with expansion of the cells, and formulation steps is carried out using a system, device, or apparatus in an integrated or self-contained system, and / or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0308] In some embodiments, immediately after the incubation, the stimulated cells can be collected for subsequent co-culture with APCs, such as in accord with methods described in Section I.C below.

[0309] In some embodiments, the stimulated cells are collected and are cryofrozen. The provision of an intermediate hold step by cryopreservation after the initial expansion phase can be used to coordinate timing with the neoepitope identification and peptide generation, such as described in Section I.A and / or the generation of APCs as described in Section I.C. In some embodiments, for cryopreservation, the stimulated cells are formulated as a composition with a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150°C, such as or about 80 °C ± 6.0 ° C.

[0310] In some embodiments, the cryopreserved cells are prepared for subsequent steps by thawing. In some cases, the cells can be ready for subsequent culturing with APCs and peptides immediately after thawing following one or more wash steps.C. Co-Culture of T cells with APCs

[0311] In some embodiments, the source of cells for expansion can be obtained from an ex vivo co-culture source of cells generated to be enriched for tumor reactive T cells. Methods for generating cells with antigen-specificity are known, see e.g. US published application No. US2017 / 0224800. Thus, embodiments, the provided methods include those in which the population of T cells containing tumor-reactive T cells, such as T cells that exhibit antigenic specificity for a tumor-associated antigen (e.g. neoantigen) or a peptide of a tumor-associated antigen, are identified or generated ex vivo. Such methods include, but are not limited to the steps of (1) enriching a population of T cells obtained from a donor subject, such as by directly selecting T cells from a biological sample, e.g. a tumor, blood, bone marrow, lymph node, thyus or other tissue or fluids; (2) stimulating the population with one or more T-cell stimulating agents, such as described above (e.g. anti-CD3 (e.g. OKT3) and anti-CD28 reagents, such as an anti-CD3 antibody (e.g. OKT3) and an anti-CD28 antibody, and one or more additional recombinant cytokine such as IL-2, IL-7, IL-21 and / or IL-15), to produce a population containing activated T cells; (3) co-culturing the population containing activated T cells in the presence of antigen presenting cells (APCs) that present one or more MHC-associated non-native peptide; and (4) enriching T cells containing an endogenous TCR that are reactive to peptides present on antigen presenting cells (APCs). In some aspects, T cells containing an endogenous TCR are enriched by separating the antigen presenting cells from the population of T cells. 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.

[0312] In particular embodiments, once the neopitopes that encode for proteins are synthesized a plurality of the synthetic peptides are contacted with antigen presenting cells under conditions to present peptides in the context of an MHC molecule and incubated with T cells from a population of T cells for recognition of the peptides presented on the APCs. In some embodiments, the synthetic peptides are pulsed into autologous or allogeneic APCs that are then cultured with patient T cells. Antigen presenting cells are used to present these peptides. T cells that recognize these peptides on the surface of the APC can then be isolated, such as by methods described below. The incubated cells can be cultured under conditions that enrich for and expand tumor-reactive T cells, i.e. T cells containing endogenous TCR that are reactive to peptides present on the APCs, in the culture. In some embodiments, the methods include culturing the T cells under conditions for expansion until a threshold amount of T cells is obtained and / or until up to 20 days after initiation of incubation. In some embodiments, of the provided methods the method can include co-culturing the T cells with the APCs over the course of several hours to days and then separating antigen presenting cells from the population of T cells for the expansion of the T cells under conditions to enrich or expand tumor-reactive T cells. In some embodiments, of the provided methods the method can include co-culturing the T cells with the APCs over the course of 1-7 days, and then separating antigen presenting cells from the population of T cells for the expansion of the T cells under conditions to enrich or expand tumor-reactive T cells. In some embodiments, the separating can include isolating or selecting reactive T cells from culture based on one or more T cell activation markers on T cells.

[0313] The method may comprise inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence. The method may comprise inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence. The APCs may include any cells which present peptide fragments of proteins in association with major histocompatibility complex (MHC) molecules on their cell surface. The APCs may include, for example, any one or more of macrophages, DCs, Langerhans cells, B-lymphocytes, and T-cells. Preferably, the APCs are DCs. By using autologous APCs from the patient, the methods may identify T cells that have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation that is presented in the context of an MHC molecule expressed by the patient. The MHC molecule can be any MHC molecule expressed by the patient including, but not limited to, MHC Class I, MHC Class II, HLA-A, HLA-B, HLA-C, HLA-DM, HLA-DO, HLA-DP, HLA-DQ, and HLA-DR molecules.

[0314] In particular embodiments, the APCs include cells that are able to present Class I and Class II restricted molecules. For example, B cells and DCs both have the ability to present MHC class I and MHC class II restricted molecules. In some embodiments, the APC cell sample includes B cells and DCs. In some embodiments, the APC cell sample is enriched for B cells, such as by selection or isolation from a primary cell sample. In some embodiments, the APC cell sample is enriched for DCs, such as by selection or isolation from a primary cell sample.

[0315] In some embodiments, the APCs express MHC class I and / or MHC class II molecules with a matched HLA from which the source of T cells has been obtained. In particular embodiments, both the APCs and T cells have been isolated from the same subject, i.e. are autologous to the cancer patient. In some embodiments, the method may comprise inducing autologous antigen presenting cells (APCs) of the patient to present the mutated amino acid sequence. By using autologous APCs from the patient, the methods may identify T cells that have antigenic specificity for a mutated amino acid sequence encoded by a cancer-specific mutation that is presented in the context of an MHC molecule expressed by the patient.

[0316] In some embodiments, the APCs are cells from a blood or apheresis sample from a subject, such as the patient. In some embodiments, the APCs include cells present in a peripheral blood mononuclear cell (PBMC) sample Typically, APCs function in a PBMC culture primarily involves monocytes and B cells. In some embodiments, a population of isolated PBMCs can be used as APCs in the provided methods. PBMCs can be obtained using standard methods such as Ficoll-Paque gradient separation. In some cases, the APCs are or include B cells that are isolated from the blood or apheresis sample or from a PBMC sample. In other cases, the APCs are or include monocytes isolated from the blood or apheresis sample or from a PBMC sample. In some aspects, the monocytes can be used as a source for preparing monocyte-derived DCs for use as APCs. In some embodiments, a source of monocyte-derived DCs (e.g. CD11c high< MHCII high< CD14 low< cells) can be generated ex vivo from isolated monocytes, by culture with GM-CSF and IL-4 for 4 to 6 days to produce monocyte-derived dendritic cells. In particular embodiments, the monocytes are isolated from PBMCs such as by CD14 selection, and then are cultured with GM-CSF and IL-4 for 4 to 6 days.

[0317] In some embodiments, the APCs are primary cells (e.g. B cells or monocyte-derived DCs) that are replication competent, for example, the cells are not subjected to irradiation, heat treatment or other method that would result in their inactivation. In particular embodiments, the provided methods do not use irradiated APCs. In some embodiments, the APCs are freshly isolated primary cells obtained from the subject, or are derived from primary cells obtained from the subject. In some embodiments, the APCs have been cryopreserved and subsequently thawed prior to the co-culture with the stimulated T cells in accord with provided methods.

[0318] In some particular embodiments, B cells are used as a source of APCs and are generated from a patient apheresis, such as an apheresis autologous to the subject from which the tumor fragments and / or T cells were obtained. In other particular embodiments, monocyte-derived dendritic cells are used as a source of APCs and are generated from monocytes from a patient apheresis, such as an apheresis autologous to the subject from which the tumor fragment and / or T cells are obtained.

[0319] In some embodiments, the isolated or generated APCs are collected and are cryofrozen. The provision of an intermediate hold step by cryopreservation after the isolation or generation of APCs can be used to coordinate timing with the neoepitope identification and peptide generation such as described in Section I.A and / or initial expansion of T cells, such as described in Section I.B. In some embodiments, for cryopreservation, the isolated or generated APCs are formulated as a composition with a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or s glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, such as ultra low temperatures, for example, storage with temperature ranges from -40 °C to -150°C, such as or about 80 °C ± 6.0 ° C.

[0320] In some embodiments, the cryopreserved cells are prepared for subsequent steps by thawing. In some cases, the cells can be ready for subsequent culturing with T cells and peptides immediately after thawing following one or more wash steps.

[0321] In particular embodiments, the methods for enriching or selecting tumor reactive cells are initiated by contacting PBMCs with the mutated amino acid sequence, such as one or more, such as a plurality of, neoepitope peptides. The PBMCs / peptides can then be cultured with stimulated T cells. The PBMCs and T cells can be obtained from the same subject.

[0322] In particular embodiments, the methods for enriching or selecting tumor reactive cells are initiated by contacting B cells with the mutated amino acid sequence, such as one or more, such as a plurality of, neoepitope peptides. The B cell / peptides can then be cultured with stimulated T cells. The B cells and T cells can be obtained from the same subject.

[0323] In particular embodiments, the methods for enriching or selecting tumor reactive cells are initiated by contacting monocyte-derived DCs with the mutated amino acid sequence, such as one or more, such as a plurality of, neoepitope peptides. The monocyte-derived Dcs / peptides can then be cultured with stimulated T cells. The monocyte-derived DCs and T cells can be obtained or derived from the same subject.

[0324] In some embodiments, the APC is an artificial antigen presenting cell (aAPC). Typically, aAPCs include features of natural APCs, including expression of an MHC molecule, stimulatory and costimulatory molecule(s), Fc receptor, adhesion molecule(s) and / or the ability to produce or secrete cytokines (e.g. IL-2). Normally, an aAPC is a cell line that lacks expression of one or more of the above, and is generated by introduction (e.g. by transfection or transduction) of one or more of the missing elements from among an MHC molecule, a low affinity Fc receptor (CD32), a high affinity Fc receptor (CD64), one or more of a co-stimulatory signal (e.g. CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, ILT3, ILT4, 3 / TR6 or a ligand of B7-H3; or an antibody that specifically binds to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, Toll ligand receptor or a ligand of CD83), a cell adhesion molecule (e.g. ICAM-1 or LFA-3) and / or a cytokine (e.g. IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, interferon-alpha (IFNα), interferon-beta (IFNβ), interferon-gamma (IFNγ), tumor necrosis factor-alpha (TNFα), tumor necrosis factor-beta (TNFβ), granulocyte macrophage colony stimulating factor (GM-CSF), and granulocyte colony stimulating factor (GCSF)). In some cases, an aAPC does not normally express an MHC molecule, but can be engineered to express an MHC molecule or, in some cases, is or can be induced to express an MHC molecule, such as by stimulation with cytokines. In some cases, aAPCs also can be loaded with a stimulatory or co-stimulatory ligand, which can include, for example, an anti-CD3 antibody, an anti-CD28 antibody or an anti-CD2 antibody. Exemplary of a cell line that can be used as a backbone for generating an aAPC is a K562 cell line or fibroblast cell line. Various aAPCs are known in the art, see e.g., U.S. Patent No. 8,722,400, published application No. US2014 / 0212446; Butler and Hirano (2014) Immunol Rev., 257(1):10. 1111 / imr.12129; Suhoshki et al. (2007) Mol. Ther., 15:981-988). In particular embodiments, the methods for enriching or selecting tumor reactive cells are initiated by contacting aAPCs with the mutated amino acid sequence, such as one or more, such as a plurality of, neoepitope peptides. The aAPC / peptides can then be cultured with stimulated T cells.

[0325] In a particular embodiment, whole exome sequencing is performed on healthy and diseased tissue to identify somatic mutations associated with the tumor. Once these mutations are identified, neoepitopes, the reactive neoantigens are then identified. Neoantigens are mutant peptides that are recognized by a patient's T cells. These neoantigens must be presented by a tumor or antigen presenting cell by the MHC complex and then be recognized by a TCR on the T cell. Once the neopitopes that encode for proteins are identified a mutation library can be generated. Long peptides can be synthesized and pulsed using electroporation into an antigen presenting cell. Long peptides can then be presented by the antigen presenting cells to be recognized by CD8 cells. Long peptides are suitable for expression by MHC class I restricted molecules for recognition by CD8 cells. Long peptides do not work for MHC class II restricted molecules for recognition by CD4 cells. MHC class II restricted molecules must be presented as a gene encoding DNA of the mutation and electroporated into the antigen presenting cell.

[0326] Inducing autologous APCs (e.g. B cells or monocyte-derived DCs) of the patient to present the mutated amino acid sequence may be carried out using various suitable methods. In an embodiment, inducing autologous APCs of the patient to present the mutated amino acid sequence (i.e. peptide neoepitope) comprises pulsing the autologous APCs with peptides comprising the mutated amino acid sequence or a pool of peptides, each peptide in the pool comprising a different mutated amino acid sequence. In some cases, the APCs are pulsed with the peptides using electroporation into an antigen presenting cell. The synthetic peptides can then be presented by the antigen presenting cells to be recognized by CD8 cells (MHC class I) or CD4 cells (MHC class II). In certain particular embodiments, synthetic peptides are generated to be suitable for expression by MHC class I restricted molecules for recognition by CD8 cells. In other particular embodiments, synthetic peptides are generated to be suitable for expression by MHC class II restricted molecules for recognition by CD4 cells.

[0327] In some embodiments, the APCs (e.g. PBMCs, B cells or monocyte-derived DCs) are contacted with a single peptide or a pool of peptides. The pool of peptide can represent many different mutated amino acid sequences, such as 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 100 peptides, or any value between any of the foregoing.

[0328] The peptides or pool of peptides are loaded onto antigen presenting cells (e.g. dendritic cells), such as by peptide pulsing, at a concentrations suitable for their presentation on the surface of a major histocompatibility complex (MHC).

[0329] In some embodiments, the peptide concentration representing an individual or single peptide can range between at or about 0.00000 1 µg / mL and at or about 10 µg / m L. In some embodiments, the peptide concentration representing an individual or single peptide can range between at or about 0.00001 µg / mL and at or about 10 µg / mL, at or about 0.00001 µg / mL and at or about 1 µg / mL, at or about 0.00001 µg / mL and at or about 0.1 µg / mL, at or about 0.00001 µg / mL and at or about 0.01 µg / mL, at or about 0.00001 µg / mL and at or about 0.001 µg / mL, at or about 0.00001 µg / mL and at or about 0.0001 µg / mL, at or about 0.0001 µg / mL and 10 µg / mL, at or about 0.0001 µg / mL and at or about 1 µg / mL, at or about 0.0001 µg / mL and at or about 0.1 µg / mL, at or about 0.0001 µg / mL and at or about 0.01 µg / mL, at or about 0.0001 µg / mL and at or about 0.001 µg / mL, at or about 0.001 µg / mL and at or about 10 µg / mL, at or about 0.001 µg / mL and at or about 1 µg / mL, at or about 0.001 µg / mL and at or about 0.1 µg / mL, at or about 0.001 µg / mL and at or about 0.01 µg / mL, at or about 0.01 µg / mL and at or about 10 µg / mL, at or about 0.01 µg / mL and at or about 1 µg / mL, at or about 0.01 µg / mL and at or about 0.1 µg / mL, at or about 0.1 µg / mL and at or about 10 µg / mL, at or about 0.1 µg / mL and at or about 1 µg / mL, or at or about 1 µg / mL and at or about 10 µg / mL. In some embodiments, the concentration representing an individual or single peptide can be at or about 0.00000 1 µg / mL, at or about 0.00001 µg / mL, at or about 0.0001 µg / mL, at or about 0.001 µg / mL, at or about 0.01 µg / mL, at or about 0.1 µg / mL, at or about 1 µg / mL, or any value between any of the foregoing.

[0330] In some embodiments, the peptides are a pool of peptides representing many different mutated amino acid sequences and the concentration on average of individual or single peptides in the pool can range between at or about 0.00000 1 µg / mL and at or about 10 µg / mL. In some embodiments, the peptides are a pool of peptides representing many different mutated amino acid sequences and the concentration on average of individual or single peptides in the pool can range between at or about 0.00001 µg / mL and at or about 10 µg / mL, at or about 0.00001 µg / mL and at or about 1 µg / mL, at or about 0.00001 µg / mL and at or about 0.1 µg / mL, at or about 0.00001 µg / mL and at or about 0.01 µg / mL, at or about 0.00001 µg / mL and at or about 0.001 µg / mL, at or about 0.00001 µg / mL and at or about 0.0001 µg / mL, at or about 0.0001 µg / mL and 10 µg / mL, at or about 0.0001 µg / mL and at or about 1 µg / mL, at or about 0.0001 µg / mL and at or about 0.1 µg / mL, at or about 0.0001 µg / mL and at or about 0.01 µg / mL, at or about 0.0001 µg / mL and at or about 0.001 µg / mL, at or about 0.001 µg / mL and at or about 10 µg / mL, at or about 0.001 µg / mL and at or about 1 µg / mL, at or about 0.001 µg / mL and at or about 0.1 µg / mL, at or about 0.001 µg / mL and at or about 0.01 µg / mL, at or about 0.01 µg / mL and at or about 10 µg / mL, at or about 0.01 µg / mL and at or about 1 µg / mL, at or about 0.01 µg / mL and at or about 0.1 µg / mL, at or about 0.1 µg / mL and at or about 10 µg / mL, at or about 0.1 µg / mL and at or about 1 µg / mL, or at or about 1 µg / mL and at or about 10 µg / mL. In some embodiments, the concentration on average of individual or single peptides in the pool can be at or about 0.00000 1 µg / mL, at or about 0.00001 µg / mL, at or about 0.0001 µg / mL, at or about 0.001 µg / mL, at or about 0.01 µg / mL, at or about 0.1 µg / mL, at or about 1 µg / mL, or any value between any of the foregoing.

[0331] In some embodiments, the concentration of individual peptides of the one or more non-native peptide is, on average, less than 0.02 µg / mL. In some embodiments, the concentration of individual peptides of the one or more non-native peptides is, on average, from at or about 0.00001 µg / mL to at or about 0.01 µg / mL, such as at or about 0.00001 µg / mL to at or about 0.005 µg / mL,...

Claims

1. A method for manufacturing tumor-reactive T cells, the method comprising: (1) culturing T cells by a process that comprises: (a) incubating a population of cells comprising T cells from a biological sample obtained from a subject that has a tumor with a first T cell stimulatory agent(s) under conditions to stimulate expansion of T cells of the population to produce a population of stimulated T cells, wherein the first T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines; (b) co-culturing the population of stimulated T cells in the presence of antigen presenting cells (APCs) under conditions in which the APCs have been induced to present one or more peptides from a tumor-associated antigen from the subject, thereby generating a population containing T cells comprising tumor reactive T cells; (c) enriching from the co-culture the population of tumor reactive T cells reactive to the one or more peptides, wherein said tumor-reactive T cells comprise an endogenous TCR that is reactive to a tumor-associated antigen, thereby producing a population of T cells enriched for tumor-reactive T cells; and (d) incubating the population of T cells enriched in tumor reactive T cells with a second T cell stimulatory agent(s) under conditions to stimulate expansion of T cells in the population, wherein the second T cell stimulatory agent(s) comprises the presence of one or more recombinant cytokines; and wherein one or more steps of the culturing is carried out 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 which is a tumor necrosis factor receptor superfamily (TNFRSF) agonist, or (iii) a checkpoint inhibitor which 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; and (2) harvesting cells produced by the method to produce a composition of expanded T cells enriched in tumor reactive T cells.

2. The method of claim 1, wherein: (1) the apoptosis inhibitor inhibits one or more of caspase 2, a caspase 8, a caspase 9, a caspase 10, a caspase 3, a caspase 6 or a caspase 7; (2) the apoptosis inhibitor is selected from the group consisting of Emricasan (IDN-6556, PF-03491390), NAIP (neuronal apoptosis inhibitory protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome binding IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7) and Ts-IAP (testis-specific IAP; BIRC8), Wedelolactone, NS3694, NSCI and Z-fluoromethyl ketone Z-VAD-FMK or a flouromethyl ketone variant thereof; (3) the apoptosis inhibitor is a pan-caspase inhibitor that inhibits activation or activity of two or more caspases; (4) the apoptosis inhibitor is Z-VAD-FMK; and / or (5) 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, optionally wherein: (a) the apoptosis inhibitor is added at a concentration of between at about 0.5 µM and at about 50 µM, between at about 0.5 µM and at about 40 µM, between at about 0.5 µM and at about 30 µM, between at about 0.5 µM and at about 20 µM, between at about 0.5 µM and at about 10 µM, between at about 0.5 µM and at about 5 µM, between at about 0.5 µM and at about 1 µM, between at about 1 µM, at about 1 µM at about 50 µM, between at about 1 µM and at about 40 µM, between at about 1 µM and at about 30 µM, between at about 1 µM and at about 20 µM, between at about 1 µM and at about 10 µM, between at about 1 µM and at about 5 µM, each inclusive, more optionally wherein the concentration is at or about 2 µM, at or about 10 µM or at or about 25 µM; or (b) the apoptosis inhibitor is added at a concentration of between at about 0.5 µg / mL and at or about 25 µg / mL, between at or about 0.5 µg / mL and at or about 10 µg / mL, between at or about 0.5 µg / mL and at or about 5 µg / mL, between at or about 0.5 µg / mL and at or about 1 µg / mL, between at or about 1 µg / mL and at or about 25 µg / mL, between at or about 1 µg / mL and at or about 10 µg / mL, between at or about 1 µg / mL and at or about 5 µg / mL, between at or about 5 µg / mL and at or about 25 µg / mL, between at or about 5 µg / mL and at or about 10 µg / mL, or between at or about 10 µg / mL and at or about 25 µg / mL, each inclusive.

3. The method of claim 1, wherein the costimulatory agonist is an antibody or antigen-binding fragment that specifically binds a TNFRSF member or is a fusion protein comprising an extracellular domain or binding portion thereof of a ligand of a TNFRSF member, more optionally wherein the TNFRSF member is selected from OX40, 4-1BB, GITR and CD27.

4. The method of claim 1, wherein: (1) the immune checkpoint is selected from PD-1 / PD-L1, optionally wherein the checkpoint inhibitor is an anti-PD-1 antibody or the checkpoint inhibitor is an anti-PDL1 antibody; (2) the immune checkpoint is OX40, optionally wherein the checkpoint inhibitor is an anti-OX40L antibody; or (3) the immune checkpoint is CTLA-4, optionally wherein the checkpoint inhibitor is an anti-CTLA-4 antibody.

5. The method of any of claims 1-4, wherein: (1) the first T cell stimulatory agent(s) comprise 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; or (2) the second T cell stimulatory agent(s) comprise 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, optionally wherein the concentration of each of the one or more recombinant cytokine individually is 100 IU / mL to 6000 IU / mL.

6. The method of any of claims 1-5, wherein the second T cell stimulatory agent(s) further comprises an anti-CD3 antibody, optionally OKT3, optionally wherein the concentration of the anti-CD3 antibody is at or about 50 ng / mL.

7. The method of any of claims 1-6, wherein the second T cell stimulating agent comprises feeder cells that are non-dividing peripheral blood mononuclear cells (PBMC), optionally wherein the feeder cells are gamma irradiated.

8. The method of any of claims 1-7, wherein the antigen presenting cells are nucleated cells such as dendritic cells, mononuclear phagocytes, B lymphocytes, endothelial cells or thymic epithelium, optionally wherein the antigen presenting cells are dendritic cells.

9. The method of any of claims 1-8, wherein: the one or more peptides comprises at least one neoepitope from tumor-associated antigens from the subject; and / or the one or more peptide comprises an individual peptide or a pool of peptides.

10. The method of any of claims 1-9, wherein the co-culture ratio of antigen presenting cells to T cells is between 20:1 and 1:1, between 15:1 and 1:1, between 10:1 and 1:1, between 5:1 and 1:1, between 2.5:1 and 1:1, between 1:20 and 1:1, between 1:15 and 1:1, between 1:10 and 1:1, between 1:5 and 1:1, or between 1:2.5 and 1:1, optionally wherein the co-culture ratio of antigen presenting cells to T cells is or is about 1:1.

11. The method of any of claims 1-10, wherein the co-culturing is for 2 hours to 24 hours, optionally wherein the co-culturing is for at or about 6 hours.

12. The method of any of claims 1-11, wherein the enriching for the tumor reactive T cells comprises selection of T cells surface positive for the one or more T cell activation markers, optionally wherein the one or more T cell activation marker is 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, more optionally wherein the one or more T cell activation marker is CD134 and / or CD137.

13. The method of any of claims 1-12, wherein: (1) one or more of the steps of the method is carried out in a closed system; (2) the incubating with the first T cell stimulatory agent(s) is for 7 to 21 days, optionally 7 to 14 days; (3) the incubating with the second T cell stimulatory agent(s) is for 7 to 21 days, optionally 7 to 14 days; and / or (4) the method comprises formulating the harvested cells with a cryoprotectant.

14. The method of any of claims 1-13, wherein: (1) the biological sample is a peripheral blood sample, a lymph node sample, or a tumor sample; (2) the biological sample is a resected tumor and the population of cells comprising T cells are one or more tumor fragments from the resected tumor; or (3) the biological sample is a resected tumor and the population of cells comprising T cells are processed as a single cell suspension by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor.

15. A composition comprising expanded T cells enriched in tumor reactive T cells for use in a method of treating a subject having a cancer, wherein the method comprises manufacturing the composition using the method of any one of claims 1-14 and administering the composition to a subject having a tumor a therapeutic dose of the composition, wherein the cells of the administered composition are autologous to the subject.