Methods for ex vivo enrichment and expansion of tumor-reactive T cells and related compositions thereof

Abstract

Provided herein are methods for producing tumor-reactive T cells, including ex vivo enrichment and expansion of cells secreting chemokine (C-X-C motif) ligand 13 (CXCL13); cells surface-positive for C-X-C chemokine receptor type 5 (CXCR5); and / or cells surface-positive for one or more of CD39, PD-1, and TIGIT. Also provided are T cell populations generated by the methods described herein, and pharmaceutical compositions thereof. TIFF2023516636000026.tif24170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 982,704, filed February 27, 2020, and entitled "METHODS FOR EX VIVO ENRICHMENT AND EXPANSION OF TUMOR REACTIVE T CELLS AND RELATED COMPOSITIONS THEREOF," the entire contents of which are incorporated by reference.

[0002] Incorporation by reference of sequence listing This application is filed with an electronic Sequence Listing. The Sequence Listing is provided as a file entitled 16517_2000840_SEQLIST.txt, created on March 1, 2021, and is 12,537 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

[0003] Field The present disclosure provides methods for producing tumor-reactive T cells, including ex vivo enrichment and expansion of cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13); cells that are surface-positive for C-X-C chemokine receptor type 5 (CXCR5); and / or cells that are surface-positive for one or more of CD39, PD-1, and TIGIT. The present disclosure also provides T cell populations generated by the methods described herein, and pharmaceutical compositions thereof. [Background technology]

[0004] background Clinical trials have demonstrated that T cells isolated from surgically resected tumors possess T cell receptors (TCRs) that recognize neoantigens, and expanding these neoantigen-reactive tumor-infiltrating lymphocyte (TIL) populations and reinfusing them into patients can sometimes result in dramatic clinical benefits. However, in cell therapy, a major obstacle to the application of such cells is the difficulty of obtaining such cells. For example, existing methods for generating TIL therapy for cancer use are time-consuming, involve small numbers of reactive cells, and are not suitable for commercial use. Improved methods for obtaining and manufacturing cell compositions containing tumor-reactive T cells for therapeutic use are needed. Embodiments that meet this need are provided herein. Summary of the Invention

[0005] overview According to certain embodiments described herein, a method for producing tumor-reactive T cells is provided. Such a method includes, but is not limited to, (a) selecting cells secreting chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells surface-positive for C-X-C chemokine receptor type 5 (CXCR5) from an input sample containing T cells from a subject with a tumor to obtain selected cells; and (b) culturing the selected cells with one or more T cell stimulants for lymphocytes under conditions to generate an expanded T cell population, thereby expanding the T cell population. Also provided herein are T cell populations generated by the methods described herein and pharmaceutical compositions thereof.

[0006] Provided herein are methods for producing tumor-reactive T cells, comprising: (a) selecting, from an input sample containing T cells from a subject with a tumor, cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells that are surface-positive for C-X-C chemokine receptor type 5 (CXCR5) to obtain selected cells from the sample; and (b) expanding the selected cells by culturing them with one or more T cell stimulants for lymphocytes under conditions to generate an expanded T cell population.

[0007] In some of any of the embodiments, the method includes selecting cells that secrete CXCL13. In some of any of the provided embodiments, the method includes selecting cells that are surface positive for CXCR5.

[0008] In some of any of the provided embodiments, the selecting step further comprises selecting cells surface positive for one or more additional markers selected from CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD36, CD38, CD30, CD154(CD40L), CD134(OX40), CD152(CTLA-4), CD160, CXCR5(CD195), CD244, CD258(LIGHT), CD256(APRIL), CD272(BTLA-4), PD-1, TIM-3, LAG-3, or TIGIT, wherein selecting cells that secrete CXCL13 and / or cells surface positive for CXCR5 and selecting cells surface positive for the one or more additional markers are performed simultaneously or sequentially in any order to obtain selected cells. In some of any of the provided embodiments, the one or additional marker is PD-1, CD39, and / or TIGIT.

[0009] In some of any of the provided embodiments, the method further comprises selecting T cells that are surface positive for a T cell marker selected from CD3, CD4, or CD8, wherein the steps of selecting cells that are surface positive for the T cell marker and selecting cells that secrete CXCL13 and / or cells that are surface positive for CXCR5 are performed simultaneously or sequentially in any order to obtain the selected cells. Optionally, selection by positive selection or negative selection.

[0010] Provided herein is a method for producing tumor-reactive T cells, comprising: (a) selecting cells that are surface-positive for activation markers PD-1, CD39, and TIGIT from an input sample comprising T cells from a subject with a tumor to obtain selected cells from the sample; and (b) performing expansion by culturing the selected cells with one or more T cell stimulants of lymphocytes under conditions to generate an expanded T cell population. Also provided herein is a method for producing tumor-reactive T cells, comprising: (a) selecting cells that are surface-positive for at least two activation markers from the group consisting of PD-1, CD39, and TIGIT from an input sample comprising T cells from a subject with a tumor to obtain selected cells from the sample; and performing expansion by culturing the selected cells with one or more T cell stimulants of lymphocytes under conditions to generate an expanded T cell population.

[0011] Provided herein is a method for producing tumor-reactive T cells, comprising: (a) selecting, from an input sample comprising T cells from a subject having a tumor, cells that are surface positive for PD-1, CD39, and TIGIT to obtain selected cells from the sample; and (b) expanding the selected cells by culturing them with one or more T cell stimuli of lymphocytes under conditions to generate an expanded T cell population. In some of any of the provided embodiments, the selecting step further comprises selecting cells surface positive for one or more additional markers selected from CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD36, CD38, CD30, CD154(CD40L), CD134(OX40), CD152(CTLA-4), CD160, CXCR5(CD195), CD244, CD258(LIGHT), CD256(APRIL), CD272(BTLA-4), PD-1, TIM-3, LAG-3, or TIGIT, wherein selecting cells surface positive for PD-1 / CD39 / TIGIT and selecting cells surface positive for the additional markers are performed simultaneously or sequentially in any order to obtain selected cells. In some of any of the provided embodiments, the selecting step further comprises selecting cells surface positive for one or more additional markers selected from CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (OX40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), PD-1, TIM-3, LAG-3, or TIGIT, wherein the step of selecting cells surface positive for the activation markers and selecting cells surface positive for the additional markers are performed simultaneously or sequentially in any order to obtain selected cells.In some of any of the provided embodiments, the selecting step further comprises selecting cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13), wherein selecting cells that are surface positive for PD-1 / CD39 / TIGIT and selecting cells that secrete CXCL13 are performed simultaneously or sequentially in any order to obtain selected cells. In some of any of the provided embodiments, the selecting step further comprises T cells that are surface positive for a T cell marker selected from CD3, CD4, or CD8, wherein selecting cells that are surface positive for the T cell marker and selecting cells that are surface positive for PD-1 / CD39 / TIGIT are performed simultaneously or sequentially in any order to obtain selected cells. In some of any of the provided embodiments, the selecting step further comprises T cells that are surface positive for a T cell marker selected from CD3, CD4, or CD8, wherein selecting cells that are surface positive for the T cell marker and selecting cells that are surface positive for an activation marker are performed simultaneously or sequentially in any order to obtain selected cells. Optionally, selection by positive or negative selection.

[0012] In some of the provided embodiments, the input sample comprising T cells is derived from peripheral blood or from a tumor. In some of the provided embodiments, the input sample comprises tumor-infiltrating lymphocytes. In some of the provided embodiments, the input sample comprising T cells is derived from a resected tumor. In some of the provided embodiments, the input sample comprising T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from a resected tumor. In some of the provided embodiments, the input sample comprising T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from a resected tumor.

[0013] In some of any of the provided embodiments, the enzymatic digestion is by incubation with collagenase, optionally with collagenase IV or collagenase I / II.

[0014] In some of the provided embodiments, the input sample contains 10 x 10 per gram of tumor sample from the subject. 6 pieces or approximately 10 x 10 6 to 100 x 10 per gram of subject-derived tumor sample 6 pieces or approximately 100 x 10 6 In some of the provided embodiments, the expanded T cell population comprises 100 T cells. In some of the provided embodiments, the expanded T cell population is for use as a therapeutic cell composition. In some of the provided embodiments, performing expansion to generate the expanded T cell population is for 7 to 35 days. In some of the provided embodiments, performing expansion to generate the expanded T cell population is for 7 to 28 days, optionally 14 to 28 days. In some of the provided embodiments, performing expansion to generate the expanded T cell population is for 7 to 21 days, optionally 7 to 14 days.

[0015] In some of any of the provided embodiments, the one or more T cell stimuli of the lymphocytes are an anti-CD3 agent and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, and IL-23. In some of any of the provided embodiments, the one or more T cell stimuli of the lymphocytes are an anti-CD3 agent (e.g., OKT3) and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and IL-35. In some of any of the provided embodiments, at least one of the one or more T cell stimuli is recombinant IL-2. In some of any of the provided embodiments, the culturing with the one or more T cell stimuli further comprises an apoptosis inhibitor. In some of any of the provided embodiments, the one or more T cell stimulators are one or more first T cell stimulators, the expanding step is a first expansion, and the method further includes performing a second expansion by culturing the first expanded T cell population with one or more second T cell stimulators under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0016] In some of the provided embodiments, the one or more T cell stimulators of the first expansion and the one or more T cell stimulators of the second expansion are the same. In some of the provided embodiments, the step of performing the first expansion is for 7 to 21 days, optionally 7 to 14 days. In some of the provided embodiments, the step of performing the second expansion is for 7 to 21 days, optionally 7 to 14 days.

[0017] In some of any of the provided methods, the one or more T cell stimulators are one or more first T cell stimulators, the expanding step is a first expansion, and the method further includes (c) co-culturing the first expanded T cell population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells comprising an endogenous T cell receptor that is reactive to a peptide-encoding mutation in the tumor, wherein the one or more non-native peptides are peptides that correspond to associated non-synonymous somatic mutations in the tumor of the subject; and (d) performing a second expansion by culturing the reactive T cell population with one or more second T cell stimulators under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0018] In some of the provided methods, the one or more T cell stimulators are one or more first T cell stimulators, the expanding step is a first expansion, and the method further includes the steps of: (c) co-culturing the first expanded T cell population in the presence of antigen-presenting cells presenting one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells comprising an endogenous T cell receptor reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-native peptides are peptides corresponding to associated non-synonymous somatic mutations in the tumor of interest; (d) selecting, from the reactive T cell population, cells positive for one or more markers associated with reactive T cells to generate an enriched population of reactive T cells comprising a native T cell receptor reactive to a peptide-encoding mutation of the tumor; and (e) performing a second expansion by culturing the enriched population of reactive T cells with one or more second T cell stimulators under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0019] In some of the provided embodiments, the one or more markers are markers of T cell exhaustion. In some of the provided embodiments, the one or more markers are cell surface CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (OX40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), PD-1, TIM-3, LAG-3, or TIGIT. In some of the provided embodiments, one marker or markers are PD-1, CD39, and TIGIT. In some of the provided embodiments, the one or more markers is secreted CXCL13.

[0020] In some of any of the provided methods, the method further includes selecting, optionally by positive or negative selection, T cells that are surface positive for a T cell marker selected from CD3, CD4, or CD8, wherein selecting cells that are surface positive for the T cell marker and selecting cells that are positive for a marker associated with reactive T cells are performed simultaneously or sequentially in any order to obtain an enriched population of reactive T cells.

[0021] Provided herein is a method for producing tumor-reactive T cells, comprising the steps of: (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample comprising T cells; (b) performing a first expansion by culturing the input sample comprising T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting, from the first expanded cell population, cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells that are surface-positive for C-X-C chemokine receptor type 5 (CXCR5) to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0022] (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the input sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting from the first expanded cell population cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells that are surface positive for C-X-C chemokine receptor type 5 (CXCR5) to generate a selected population; (d) selecting T cells that contain endogenous T cell receptors that are reactive to a peptide-encoding mutation in the tumor; Provided herein is a method for producing tumor-reactive T cells, comprising: (a) co-culturing the selected population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to generate a tumor-reactive T cell population containing the selected population, wherein the one or more non-native peptides are peptides that correspond to associated non-synonymous somatic mutations in the tumor of interest; and (b) performing a second expansion by culturing the reactive T cell population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0023] (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the input sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) co-culturing the first expanded cell population in the presence of antigen-presenting cells that present one or more non-natural peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells that contain endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-natural peptides are responsive to a target protein. (d) selecting, from the reactive T cell population, cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells that are surface-positive for C-X-C chemokine receptor type 5 (CXCR5) to generate a selected population; and (e) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0024] In some of the provided embodiments, the method includes selecting cells that secrete CXCL13. In some of the provided embodiments, the method includes selecting cells that are surface positive for CXCR5.

[0025] In some of any of the provided embodiments, the selecting step further comprises selecting cells surface positive for one or more additional markers selected from CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD36, CD38, CD30, CD154(CD40L), CD134(OX40), CD152(CTLA-4), CD160, CXCR5(CD195), CD244, CD258(LIGHT), CD256(APRIL), CD272(BTLA-4), PD-1, TIM-3, LAG-3, or TIGIT, wherein selecting for cells secreting CXCL13 and / or surface positive for CXCR5 and selecting for cells surface positive for the one or more additional markers are performed simultaneously or sequentially in any order to generate the selected population. In some of the embodiments provided, the one or more additional markers are PD-1, CD39, and / or TIGIT. In some of the embodiments provided, the one or more additional markers are PD-1, CD39, and TIGIT.

[0026] In some of any of the provided methods, the method further includes selecting, optionally by positive or negative selection, T cells that are surface positive for a T cell marker selected from CD3, CD4, or CD8, wherein selecting cells that are surface positive for the T cell marker and selecting cells that secrete CXCL13 and / or cells that are surface positive for CXCR5 are performed simultaneously or sequentially in any order to generate the selected cell population.

[0027] Provided herein is a method for producing tumor-reactive T cells, comprising the steps of: (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample comprising T cells; (b) performing a first expansion by culturing the sample comprising T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells from the first expanded cell population that are surface positive for activation markers PD-1, CD39, and TIGIT to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0028] Provided herein is a method for producing tumor-reactive T cells, comprising the steps of: (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample comprising T cells; (b) performing a first expansion by culturing the sample comprising T cells with one or more first T cell stimulators of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells from the first expanded cell population that are surface positive for at least two activation markers from the group consisting of PD-1, CD39, and TIGIT to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimulators under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0029] (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells from the first expanded cell population that are surface positive for activation markers PD-1, CD39, and TIGIT to generate a selected cell population; (d) performing a primary cell expansion to generate a reactive T cell population comprising T cells that contain endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor. Provided herein is a method for producing tumor-reactive T cells, comprising the steps of: (a) co-culturing a selected cell population in the presence of antigen-presenting cells presenting one or more non-native peptides on the histocompatibility complex (MHC), wherein the one or more non-native peptides are peptides corresponding to associated non-synonymous somatic mutations in the tumor of the subject; and (b) performing a second expansion by culturing the reactive T cell population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0030] (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) co-culturing the first expanded cell population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells that contain endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor, wherein the one or more Provided herein is a method for producing tumor-reactive T cells, comprising the steps of: (d) selecting, from the reactive T cell population, cells that are surface positive for activation markers PD-1, CD39, and TIGIT to generate a selected cell population; and (e) performing a second expansion by culturing the selected cell population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

[0031] In some of any of the provided embodiments, the selecting step further comprises selecting cells surface positive for one or more additional markers selected from CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (OX40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3, or LAG-3, wherein the selecting cells surface positive for PD-1 / CD39 / TIGIT and the selecting cells surface positive for the additional markers are performed simultaneously or sequentially in any order to generate the selected cell population. In some of any of the provided embodiments, the selecting step further comprises selecting cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13), and wherein selecting cells that are surface positive for PD-1 / CD39 / TIGIT and selecting cells that are surface positive for the additional marker are performed simultaneously or sequentially in any order to generate the selected cell population. In some of any of the provided embodiments, the selecting step further comprises selecting cells surface positive for one or more additional markers selected from CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (OX40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3, or LAG-3, wherein the step of selecting cells surface positive for the activation marker and the step of selecting cells surface positive for the additional marker are performed simultaneously or sequentially in any order to generate the selected cell population.

[0032] In some of any of the provided embodiments, the selecting step further comprises selecting cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13), and the selecting for cells that are surface positive for PD-1 / CD39 / TIGIT and the selecting for cells that are surface positive for the additional marker are performed simultaneously or sequentially in any order to generate a selected cell population. In some of the provided methods, the method further comprises selecting, optionally by positive or negative selection, T cells that are surface positive for a T cell marker selected from CD3, CD4, or CD8, and the selecting for cells that are surface positive for the T cell marker and the selecting for cells that are surface positive for PD-1 / CD39 / TIGIT are performed simultaneously or sequentially in any order to generate a selected cell population.

[0033] In some of the provided embodiments, the biological sample is a peripheral blood sample or a tumor sample. In some of the provided embodiments, the input sample containing T cells comprises tumor-infiltrating lymphocytes (TILs).

[0034] In some of the provided embodiments, the input sample containing T cells is derived from a resected tumor. In some of the provided embodiments, the one or more tumor fragments are 1-8 mm in diameter. In some of the provided embodiments, the one or more tumor fragments are 2 cm or larger. 2 Approximately one tumor fragment per tumor is seeded for the first expansion.

[0035] In some of the provided embodiments, the input sample comprising T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from a resected tumor. In some of the provided embodiments, the input sample comprising T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from a resected tumor. In some of the provided embodiments, the enzymatic digestion is by incubation with collagenase, optionally with collagenase IV or collagenase I / II.

[0036] In some of the provided embodiments, the input sample contains 10 x 10 per gram of tumor sample from the subject. 6 pieces or approximately 10 x 10 6 to 100 x 10 per gram of subject-derived tumor sample 6 pieces or approximately 100 x 10 6 Contains T cells.

[0037] In some of the provided embodiments, the input sample containing T cells is 2 cm 2 Approximately 5 x 10 per 5 From 2 x 10 6 pieces or approximately 2 x 10 6 Total cells are seeded for expansion.

[0038] In some of the provided embodiments, the step of performing the first expansion is for 1 to 14 days. In some of the provided embodiments, the step of performing the first expansion is for 1 or about 1 day, 2 or about 2 days, 3 or about 3 days, 4 or about 4 days, 5 or about 5 days, 6 or about 6 days, 7 or about 7 days, 8 or about 8 days, 9 or about 9 days, 10 or about 10 days, 11 or about 11 days, 12 or about 12 days, 13 or about 13 days, or 14 or about 14 days. In some of the provided embodiments, the step of performing the second expansion is for 7 to 35 days. In some of the provided embodiments, the step of performing the second expansion is for 7 to 21 days, optionally 7 to 14 days.

[0039] In some of any of the provided embodiments, the one or more T cell stimulators of the first expansion and the one or more T cell stimulators of the second expansion are the same.

[0040] In some of any of the provided embodiments, the one or more first T cell stimulators of lymphocytes are an anti-CD3 agent and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, and IL-23. In some of any of the provided embodiments, the one or more first T cell stimulators of lymphocytes for the first expansion are a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and IL-35. In some of any of the provided embodiments, at least one of the one or more first T cell stimulators is recombinant IL-2.

[0041] In some of any of the provided embodiments, the one or more first T cell stimulators include an anti-CD3 antibody, optionally OKT3. In some of the provided embodiments, the one or more first T cell stimulators do not include an anti-CD3 antibody. Optionally, the concentration of the anti-CD3 antibody is 50 ng / mL or about 50 ng / mL. In some of the provided embodiments, the one or more first T cell stimulators further include an apoptosis inhibitor. In some of the provided embodiments, the culture with the one or more first T cell stimulators further includes an apoptosis inhibitor. In some of the provided embodiments, the one or more second T cell stimulators of the lymphocytes are an anti-CD3 agent and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, and IL-23. In some of any of the provided embodiments, the one or more second T cell stimulators of lymphocytes are anti-CD3 agents and / or recombinant cytokines selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and IL-35.

[0042] In some of the provided embodiments, at least one of the one or more second T cell stimulators is recombinant IL-2. In some of the provided embodiments, the concentration of the recombinant IL-2 is between 100 IU / mL and 6000 IU / mL. In some of the provided embodiments, the concentration of the recombinant IL-2 is between 300 IU / mL and 1000 IU / mL, optionally, the concentration of the recombinant IL-2 is 300 IU / mL or about 300 IU / mL. In some of the provided embodiments, the concentration of the recombinant IL-2 is 1000 IU / mL or about 1000 IU / mL.

[0043] In some of any of the provided embodiments, the one or more second T cell stimulators comprise an anti-CD3 antibody, optionally OKT3, and optionally, the concentration of the anti-CD3 antibody is at or about 50 ng / mL.

[0044] In some of the provided embodiments, the one or more second T cell stimulators further comprise an apoptosis inhibitor. In some of the provided embodiments, the culturing with the one or more second T cell stimulators further comprises an apoptosis inhibitor. In some of the provided embodiments, the apoptosis inhibitor reduces apoptosis induced by CD95 (Fas), and optionally, the apoptosis inhibitor specifically binds to CD95 (Fas) or a CD95 ligand (Fas ligand). In some of the provided embodiments, the apoptosis inhibitor is an antibody or antigen-binding fragment. Optionally, the apoptosis inhibitor is an anti-Fas antibody or an anti-Fas ligand antibody. In some of the provided embodiments, the apoptosis inhibitor is a fusion protein comprising the extracellular domain of CD95 (Fas) or a specific-binding fragment thereof that binds to a CD95 ligand (Fas ligand) fused to an Fc immunoglobulin domain. Optionally, the apoptosis inhibitor is APG101 or CAN008. In some of the provided embodiments, the apoptosis inhibitor inhibits the activation or activity of a caspase, and optionally the caspase is caspase 2, caspase 8, caspase 9, caspase 10, caspase 3, caspase 6, or caspase 7, and optionally the caspase is caspase 3. In some of the provided embodiments, the apoptosis inhibitor is selected from the group consisting of NAIP (neuronal inhibitor of apoptosis protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome-linked IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7), and Ts-IAP (testis-specific IAP; BIRC8). In some of the provided embodiments, the apoptosis inhibitor is emericasan.In some of the provided embodiments, the apoptosis inhibitor is selected from the group consisting of emricasan (IDN-6556, PF-03491390), NAIP (neuronal inhibitor of apoptosis protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitors of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome-linked IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7), and Ts-IAP (testis-specific IAP; BIRC8), wedelolactone, NS3694, NSCI, and Z-fluoromethylketone Z-VAD-FMK, or a fluoromethylketone variant thereof. In some of the provided embodiments, the apoptosis inhibitor is a pan-caspase inhibitor that inhibits the activation or activity of two or more caspases. In some of any of the provided embodiments, the apoptosis inhibitor is Z-VAD-FMK, Z-FA-FMK, Z-VAD(OH)-FMK, Z-DEVD-FMK, Z-VAD(OM2)-FMK, or Z-VDVAD-FMK.

[0045] In some of any of the provided embodiments, the concentration of the apoptosis inhibitor is from 0.5 μM and about 0.5 μM to 50 μM or about 50 μM, 0.5 μM or about 0.5 μM to 25 μM or about 25 μM, 0.5 μM or about 0.5 μM to 10 μM or about 10 μM, 0.5 μM or about 0.5 μM to 5 μM or about 5 μM, 0.5 μM or about 0.5 μM to 1 μM or about 1 μM, 1 μM or about 1 μM to 100 μM or about 100 μM, 1 μM or about 1 μM to 50 μM or about 50 μM, 1 μM or about 1 μM to 25 μM or about 25 μM, 1 μM or about 1 μM to 10 μM or about 10 μM, 1 μM or about 1 μM to 5 μM or or about 5 μM, 5 μM or about 5 μM to 100 μM or about 100 μM, 5 μM or about 5 μM to 50 μM or about 50 μM, 5 μM or about 5 μM to 25 μM or about 25 μM, 5 μM or about 5 μM to 10 μM or about 10 μM, 10 μM or about 10 μM to 100 μM or about 100 μM, 10 μM or about 10 μM to 50 μM or about 50 μM, 10 μM or about 10 μM to 25 μM or about 25 μM, 25 μM or about 25 μM to 100 μM or about 100 μM, 25 μM or about 25 μM to 50 μM or about 50 μM, or 50 μM or about 50 μM to 100 μM or about 100 μM, inclusive.

[0046] In some of the provided embodiments, the antigen-presenting cells are dendritic cells, mononuclear phagocytes, B lymphocytes, endothelial cells, or thymic epithelium. In some of the provided embodiments, the antigen-presenting cells are dendritic cells. In some of the provided embodiments, the antigen-presenting cells are autologous to the subject.

[0047] In some of the provided embodiments, the one or more non-natural peptides comprise individual peptides or peptide pools. In some of the provided embodiments, the one or more non-natural peptides are loaded onto antigen-presenting cells by tandem transfection of in vitro transcribed synthetic minigene constructs encoding the one or more non-natural peptides, and the transcription minigene constructs generate the individual peptides. In some of the provided embodiments, the one or more non-natural peptides are loaded onto antigen-presenting cells by peptide pulsing, optionally by electroporation. In some of the provided embodiments, the one or more non-natural peptides are 5-30 amino acids in length, optionally 12-25 amino acids in length, optionally 25 amino acids in length, or about 25 amino acids in length.

[0048] In some of any of the provided embodiments, the one or more non-natural peptides are a peptide pool and the concentration of the peptides in the peptide pool for the peptide pulse is from 0.001 μg / mL or about 0.001 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL to 40 μg / mL or about 40 μg / mL, 0.1 μg / mL or about 0.1 μg / mL to 40 μg / mL or about 40 μg / mL, 1 μg / mL or about 1 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL or about 0.01 μg / mL to 10 μg / mL or about 10 μg / mL, or 1 μg / mL or about 1 μg / mL to 10 μg / mL or about 10 μg / mL, or the one or more non-natural peptides are individual peptides and the concentration of the individual peptides for the peptide pulse is from 0.01 μg / mL to 40 μg / mL or about 40 μg / mL, ...10 μg / mL or about 1 μg / mL to 10 μg / mL or about 10 μg / mL, or The concentration of the peptide may be from 0.00001 μg / mL or about 0.00001 μg / mL to 1 μg / mL or about 1 μg / mL, from 0.00001 μg / mL or about 0.00001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, from 0.00001 μg / mL or about 0.00001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL, from 0.0001 μg / mL or about 0.0001 μg / mL to 1 μg / mL or about 1 μg / mL, 0.0001 μg / mL or about 0.0001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, 0.0001 μg / mL or about 0.0001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, or 0.0001 μg / mL or about 0.0001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL.

[0049] In some of the provided embodiments, the concentration of individual peptides of the one or more non-natural peptides is, on average, from at or about 0.00001 μg / mL to at or about 0.00001 μg / mL to at or about 0.01 μg / mL. In some of the provided embodiments, the concentration of individual peptides of the one or more non-natural peptides is, on average, from at or about 0.0001 μg / mL to at or about 0.001 μg / mL.

[0050] In some of the provided embodiments, the co-culture ratio of antigen-presenting cells to T cells is 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 5:1 to 1:1, 2.5:1 to 1:1, 1:20 to 1:1, 1:15 to 1:1, 1:10 to 1:1, 1:5 to 1:1, or 1:2.5 to 1:1. In some of the provided embodiments, the co-culture ratio of dendritic cells to T cells is 5:1 to 1:5, or 3:1 to 1:3, optionally 1:1 or about 1:1. In some of the provided embodiments, the co-culture ratio of antigen-presenting cells to T cells is 1:1 or about 1:1.

[0051] In some of any of the provided embodiments, the co-culturing step lasts for between 2 hours and 24 hours. In some of the provided embodiments, the co-culturing step lasts for at or about 6 hours.

[0052] In some of the provided embodiments, the step of selecting cells is performed using a fluorescence-based cell sorter. In some of the provided embodiments, the fluorescence-based cell sorter is an automated high-throughput flow cytometry sorter. Optionally, an FX500 cell sorter or a Miltenyi Tyto cell sorter. In some of the provided embodiments, the selection is performed using one run, two runs, three runs, or four runs of the fluorescence-based cell sorter. In some of the provided embodiments, the selection is performed using a disposable fluorescence-based fluidic cell sorter at a rate of 10,000 to 100,000 cells / second.

[0053] In some of the provided embodiments, the expansion culture is for 7 to 35 days. In some of the provided embodiments, the expansion culture is for 7 to 21 days, optionally 7 to 14 days. In some of the provided embodiments, the culture is performed in a closed system. In some of the provided embodiments, the first expansion culture is for 7 to 21 days, optionally 7 to 14 days.

[0054] In some of the provided embodiments, the culturing for the first expansion is carried out in a closed system using a gas-permeable culture vessel. In some of the provided embodiments, the culturing for the first expansion is carried out in a closed system using a bioreactor. In some of the provided embodiments, the step of carrying out the first expansion is carried out in a closed system using a bioreactor. In some of the provided embodiments, the culturing for the second expansion is for 7 to 21 days, optionally 7 to 14 days. In some of the provided embodiments, the culturing for the second expansion is carried out in a gas-permeable culture vessel. In some of the provided embodiments, the culturing for the second expansion is carried out using a bioreactor. In some of the provided embodiments, the run for the second expansion is carried out in a gas-permeable culture vessel. In some of the provided embodiments, the run for the second expansion is carried out using a bioreactor.

[0055] In some of the provided embodiments, the tumor is an epithelial cancer tumor. In some of the provided embodiments, the tumor is a melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, gastric cancer, or uterine cancer tumor. In some of the provided embodiments, the tumor is melanoma. In some of the provided embodiments, the tumor is colorectal cancer (CRC). In some of the provided embodiments, the tumor is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer tumor, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer.

[0056] In some of any of the provided embodiments, the method results in a fold expansion of T cells or a fold expansion of tumor-reactive T cells from the input sample that is at least or at least about 2-fold, at least or at least about 5-fold, at least or at least about 10-fold, at least or at least about 25-fold, at least or at least about 25-fold, at least 50-fold, at least 100-fold, at least 250-fold, at least 500-fold, at least 750-fold, at least 1000-fold, at least 1500-fold, at least 2000-fold, at least 2500-fold, or at least about 2500-fold, or at least 3000-fold.

[0057] In some of any of the provided embodiments, the composition of tumor-reactive cells produced by the method can produce IFNγ at a concentration of greater than or greater than about 30 pg / mL, optionally greater than or greater than about 60 pg / mL, after antigen-specific stimulation.

[0058] In some of the provided methods, the method further comprises harvesting the cells produced by the method for formulation as a therapeutic composition. In some of the provided methods, the method comprises formulating the harvested cells with a cryoprotectant.

[0059] Provided herein are compositions comprising tumor-reactive T cells produced by any of the provided methods. In some of the provided embodiments, the compositions comprise a cryoprotectant.

[0060] In some of the provided embodiments, the T cells are CD3+ T cells or comprise CD4+ T cells and / or CD8+ T cells. In some of the provided embodiments, the T cells comprise CD4+ T cells and CD8+ T cells, and the ratio of CD8+ T cells to CD4+ T cells is from at or about 1:100 to 100:1 or about 100:1, 1:50 or about 1:50 to 50:1 or about 50:1, 1:25 or about 1:25 to 25:1 or about 25:1, 1:10 or about 1:10 to 10:1 or about 10:1, 1:5 or about 1:5 to 5:1 or about 5:1, or 1:2.5 or about 1:2.5 to 2.5:1 or about 2.5:1.

[0061] In some of any of the provided embodiments, the number of tumor-reactive T cells or the number of live cells thereof in the composition is 0.5×10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 0.5×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 60 x 10 8or approximately 60 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 3.5 x 10 8 or approximately 3.5 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 pieces, 1×10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 1×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 3.5 x 10 8 or approximately 3.5 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 108 From 50 x 1 09 Pieces or approximately 50 x 1 09 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 15×108 or approximately 15×108, 3.5×10 8 pieces or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 60×108 or approximately 60×108, 8×10 8 pieces or approximately 8 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 50 x 10 9 or approximately 50 x 10 9pieces, 15×10 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 60×10 8 or approximately 60 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 60×10 8 or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 60×10 8 or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 pieces, 12 x 10 pieces or approximately 12 x 10 pieces, to 30 x 10 9 pieces or approximately 30 x 10 9 pieces, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 or approximately 60 x 10 9 (inclusive).

[0062] In some of any of the provided embodiments, the number of tumor-reactive T cells or the number of live cells thereof in the composition is at least 5×10 8 or at least about 5 x 10 8 In some of any of the provided embodiments, the number of tumor-reactive T cells or the number of live cells thereof in the composition is at least 1 x 109 or at least about 1 x 10 9 In some of any of the provided embodiments, the number of tumor-reactive T cells or the number of live cells thereof in the composition is at least 10 x 10 9 or at least about 10 x 10 9 In some of the provided aspects, the provided compositions comprise a pharmaceutically acceptable excipient. Provided herein is a method of treatment comprising administering any of the provided compositions to a subject with cancer. In some of the provided aspects, the cells of the administered composition are autologous to the subject.

[0063] In some of any of the provided embodiments, the therapeutically effective dose is 1×10 8 ~10×10 9 In some of the provided embodiments, the therapeutically effective dose is 5×10 T cells or live cells thereof. 8 ~10×10 9 In some of the provided embodiments, the therapeutically effective dose is 5×10 T cells or live cells thereof. 8 ~1×10 9 The T cells or live cells thereof.

[0064] In some of the provided embodiments, the cancer is an epithelial cancer. In some of the provided embodiments, the cancer is melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung cancer, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer. In some of the provided embodiments, the cancer is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer.

[0065] Provided herein are compositions for use in treating a subject with cancer. Also provided herein is the use of any of the provided compositions for the manufacture of a medicament for treating a subject with cancer.

[0066] In some of the provided embodiments, the cells of the administered composition are autologous to the subject.

[0067] In some of any of the provided embodiments, the therapeutically effective dose is 1×10 8 ~10×10 9 In some of the provided embodiments, the therapeutically effective dose is 5×10 T cells or live cells thereof. 8 ~10×10 9 In some of the provided embodiments, the therapeutically effective dose is 5×10 T cells or live cells thereof. 8 ~1×10 9 The T cells or live cells thereof.

[0068] In some of the provided embodiments, the cancer is an epithelial cancer. In some of the provided embodiments, the cancer is melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung cancer, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer. In some of the provided embodiments, the cancer is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer. [The present invention 1001] a. selecting cells from an input sample comprising T cells from a tumor-bearing subject that are surface positive for activation markers PD-1, CD39, and TIGIT to obtain selected cells from the sample; and b. Expanding by culturing the selected cells with one or more T cell stimulants of lymphocytes under conditions to produce an expanded T cell population. A method for producing tumor-reactive T cells, comprising: [The present invention 1002] a. selecting cells from an input sample comprising T cells from a subject with a tumor that are surface positive for at least two activation markers selected from the group consisting of PD-1, CD39, and TIGIT to obtain selected cells from the sample; and b. Expanding by culturing the selected cells with one or more T cell stimulants of lymphocytes under conditions to produce an expanded T cell population. A method for producing tumor-reactive T cells, comprising: [The present invention 1003] 1001. The method of claim 1001, wherein said selecting step further comprises selecting cells surface-positive for one or more additional markers selected from CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (OX40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), PD-1, TIM-3, LAG-3, or TIGIT, and wherein the step of selecting cells surface-positive for said activation markers and the step of selecting cells surface-positive for said additional markers are performed simultaneously or sequentially in any order to obtain said selected cells. [The present invention 1004] Any of the methods of inventions 1001 to 1003, optionally further comprising the step of selecting, by positive or negative selection, T cells that are surface-positive for a T cell marker selected from CD3, CD4, or CD8, wherein the step of selecting cells that are surface-positive for said T cell marker and the step of selecting cells that are surface-positive for said activation marker are performed simultaneously or sequentially in any order to obtain said selected cells. [The present invention 1005] 1012. The method of any one of claims 1001 to 1011, wherein said input sample comprising T cells is derived from peripheral blood or from a tumor. [The present invention 1006] 1006. The method of any one of claims 1001 to 1005, wherein said input sample comprises tumor-infiltrating lymphocytes. [The present invention 1007] 1007. The method of any one of claims 1001 to 1006, wherein said input sample comprising T cells is derived from a resected tumor. [The present invention 1008] 1007. The method of claim 1007, wherein said input sample containing T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from said resected tumor. [The present invention 1009] The method of claim 1007 or claim 1008, wherein said input sample comprising T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from said resected tumor. [The present invention 1010] 1009. The method of any of claims 1008 and 1009, wherein said enzymatic digestion is by incubation with collagenase, optionally by incubation with collagenase IV or collagenase I / II. [The present invention 1011] The input sample contains 10 x 10 per gram of tumor sample from the subject. 6 pieces or approximately 10 x 10 6 to 100 x 10 per gram of tumor sample from the subject 6 pieces or approximately 100 x 10 6 The method of any one of 1001 to 1010, comprising T cells. [The present invention 1012] The method of any one of claims 1001 to 1011, wherein the expanded T cell population is for use as a therapeutic cell composition. [The present invention 1013] 13. The method of any of claims 1001 to 1012, wherein said expanding to generate said expanded T cell population takes between 7 and 35 days. [The present invention 1014] Any of the methods of claims 1001 to 1013, wherein said expanding to generate said expanded T cell population is for 7 to 28 days, optionally 14 to 28 days. [The present invention 1015] 14. The method of any of claims 1001 to 1013, wherein said expanding to produce said expanded T cell population is for 7 to 21 days, optionally 7 to 14 days. [The present invention 1016] Any of the methods of inventions 1001 to 1015, wherein the one or more T cell stimulators are one or more first T cell stimulators, the step of performing the expansion is a first expansion, and the method further comprises the step of performing a second expansion by culturing the first expanded T cell population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, and the second expanded T cell population is for use as a therapeutic cell composition. [The present invention 1017] 1016. The method of claim 1016, wherein said one or more T cell stimulators of said first expansion and said one or more T cell stimulators of said second expansion are the same. [The present invention 1018] 1018. The method of claim 1016 or 1017, wherein said step of performing the first expansion lasts for 7 to 21 days, optionally for 7 to 14 days. [The present invention 1019] 19. The method of any one of claims 1016 to 1018, wherein the second expansion step is for 7 to 21 days, optionally 7 to 14 days. [The present invention 1020] Any of the methods of claims 1001 to 1019, wherein said one or more T cell stimulants of lymphocytes are an anti-CD3 substance (e.g., OKT3) and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and IL-35. [The present invention 1021] The method of any of claims 1001 to 1020, wherein at least one of said one or more T cell stimulatory agents is recombinant IL-2. [The present invention 1022] The method of any of claims 1001 to 1021, wherein said culturing with one or more T cell stimulants further comprises an apoptosis inhibitor. [The present invention 1023] a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. selecting cells from the first expanded cell population that are surface positive for activation markers PD-1, CD39, and TIGIT to generate a selected population; and d. performing a second expansion by culturing the selected population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. A method for producing tumor-reactive T cells, comprising: [The present invention 1024] a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. selecting cells from the first expanded cell population that are surface positive for at least two activation markers from the group consisting of PD-1, CD39, and TIGIT to generate a selected population; and d. performing a second expansion by culturing the selected population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. A method for producing tumor-reactive T cells, comprising: [The present invention 1025] a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. selecting cells from the first expanded cell population that are surface positive for activation markers PD-1, CD39, and TIGIT to generate a selected cell population; and d. co-culturing the selected cell population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells that comprise endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-native peptides are peptides that correspond to an associated non-synonymous somatic mutation in the tumor of interest; e. performing a second expansion by culturing the reactive T cell population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. A method for producing tumor-reactive T cells, comprising: [The present invention 1026] a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. co-culturing the first expanded cell population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells that comprise an endogenous T cell receptor that is reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-native peptides are peptides that correspond to an associated non-synonymous somatic mutation in the tumor of interest; d. selecting cells from the reactive T cell population that are surface positive for the activation markers PD-1, CD39, and TIGIT to generate a selected cell population; and e. performing a second expansion by culturing the selected cell population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. A method for producing tumor-reactive T cells, comprising: [The present invention 1027] 1027. The method of any of claims 1023 to 1026, wherein the selecting step further comprises selecting cells surface-positive for one or more additional markers selected from CD107a, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (OX40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), TIM-3, or LAG-3, wherein the step of selecting cells surface-positive for said activation markers and the step of selecting cells surface-positive for said additional markers are performed simultaneously or sequentially in any order to generate the selected cell population. [The present invention 1028] Any of the methods of inventions 1023 to 1027, optionally further comprising the step of selecting, by positive or negative selection, T cells that are surface positive for a T cell marker selected from CD3, CD4, or CD8, wherein the steps of selecting cells that are surface positive for said T cell marker and selecting cells that are surface positive for said activation marker are performed simultaneously or sequentially in any order to generate said selected cell population. [The present invention 1029] The method of any one of claims 1023 to 1028, wherein said biological sample is a peripheral blood sample or a tumor sample. [The present invention 1030] 1029. The method of any one of claims 1023 to 1029, wherein said input sample comprising T cells comprises tumor infiltrating lymphocytes (TILs). [The present invention 1031] 1030. The method of any one of claims 1023 to 1030, wherein said input sample comprising T cells is derived from a resected tumor. [The present invention 1032] 1032. The method of any of claims 1023 to 1031, wherein said biological sample is a resected tumor from said subject, and said input sample comprising T cells is one or more tumor fragments from said resected tumor. [The present invention 1033] 1033. The method of claim 1032, wherein said one or more tumor fragments are 1 to 8 mm in diameter. [The present invention 1034] 1034. The method of claim 1032 or 1033, wherein said one or more tumor fragments are seeded for said first expansion at about 1 tumor fragment per 2 cm2. [This invention 1035] 1032. The method of any of claims 1023 to 1031, wherein the input sample containing T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor. [The present invention 1036] 1032. The method of any of claims 1023 to 1031, wherein said input sample containing T cells is a single cell suspension processed by homogenization and enzymatic digestion of one or more tumor fragments from a resected tumor. [This invention 1037] 1037. The method of claim 1035 and claim 1036, wherein said enzymatic digestion is by incubation with collagenase, optionally by incubation with collagenase IV or collagenase I / II. [The present invention 1038] The input sample contains 10 x 10 per gram of tumor sample from the subject. 6 pieces or approximately 10 x 10 6 to 100 x 10 per gram of tumor sample from the subject 6 pieces or approximately 100 x 10 6 The method of any one of claims 1023 to 1037, comprising T cells. [This invention 1039] 10. The method of any of claims 10 to 10, wherein the input sample containing T cells is seeded for expansion at about 5 x 10 to 2 x 10 total cells per 2 cm, or at about 2 x 10 total cells per 2 cm. [The present invention 1040] 1039. The method of any one of claims 1023 to 1039, wherein the step of performing the first expansion lasts for 1 to 14 days. [The present invention 1041] 10. The method of any of claims 1023 to 1040, wherein the step of performing the first expansion lasts for 1 day or about 1 day, 2 days or about 2 days, 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, 6 days or about 6 days, 7 days or about 7 days, 8 days or about 8 days, 9 days or about 9 days, 10 days or about 10 days, 11 days or about 11 days, 12 days or about 12 days, 13 days or about 13 days, or 14 days or about 14 days. [The present invention 1042] 1042. The method of any one of claims 1023 to 1041, wherein the step of carrying out the second expansion lasts for 7 to 35 days. [This invention 1043] 1043. The method of any one of claims 1023 to 1042, wherein the second expansion step is for 7 to 21 days, optionally 7 to 14 days. [This invention 1044] The method of any of claims 1023 to 1043, wherein said one or more T cell stimulators of said first expansion and said one or more T cell stimulators of said second expansion are the same. [This invention 1045] 104. The method of any of claims 1023 to 1044, wherein said one or more first T cell stimuli of lymphocytes for said first expansion are recombinant cytokines selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and IL-35. [The present invention 1046] 1046. The method of any of claims 1023 to 1045, wherein at least one of said one or more first T cell stimulatory agents is recombinant IL-2. [This invention 1047] 1047. The method of any of claims 1023 to 1046, wherein said one or more first T cell stimulants does not include an anti-CD3 antibody (e.g., OKT3). [This invention 1048] 1048. The method of any of claims 1023 to 1047, wherein said culturing with said one or more first T cell stimulants further comprises an apoptosis inhibitor. [This invention 1049] 9. The method of any of claims 1023 to 1048, wherein said one or more second T cell stimuli of lymphocytes is an anti-CD3 substance and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and IL-35. [The present invention 1050] 1049. The method of any of claims 1023 to 1049, wherein at least one of said one or more second T cell stimulatory agents is recombinant IL-2. [This invention 1051] Any of the methods of inventions 1020, 1021, 1045, 1046, 1049, and 1050, wherein the concentration of recombinant IL-2 is 100 IU / mL to 6000 IU / mL. [This invention 1052] 10. The method of any of claims 1020, 1021, 1045, 1046, and 1049-1051, wherein the concentration of the recombinant IL-2 is between 300 IU / mL and 1000 IU / mL, and optionally, the concentration of the recombinant IL-2 is 300 IU / mL or about 300 IU / mL. [This invention 1053] The method of any of claims 1020, 1021, 1045, 1046, and 1049 to 1052, wherein the concentration of recombinant IL-2 is 300 IU / mL or about 300 IU / mL. [This invention 1054] The method of any of claims 1020, 1021, 1045, 1046, and 1049 to 1052, wherein the concentration of recombinant IL-2 is 1000 IU / mL or about 1000 IU / mL. [This invention 1055] 105. The method of any of claims 1023 to 1054, wherein the one or more second T cell stimulants comprise an anti-CD3 antibody, optionally OKT3, and optionally, the concentration of the anti-CD3 antibody is 50 ng / mL or about 50 ng / mL. [This invention 1056] 1056. The method of any of claims 1023 to 1055, wherein said culturing with said one or more second T cell stimulants further comprises an apoptosis inhibitor. [This invention 1057] The method of claim 1022, claim 1048, or claim 1056, wherein the apoptosis inhibitor reduces apoptosis induced by CD95 (Fas), and optionally, the apoptosis inhibitor specifically binds to CD95 (Fas) or a CD95 ligand (Fas ligand). [This invention 1058] 1057. The method of claim 1057, wherein said apoptosis inhibitor is an antibody or antigen-binding fragment, and optionally, said apoptosis inhibitor is an anti-Fas antibody or an anti-Fas ligand antibody. [This invention 1059] 1057. The method of claim 1057, wherein the apoptosis inhibitor is a fusion protein comprising the extracellular domain of CD95 (Fas) or a specific binding fragment thereof that binds to a CD95 ligand (Fas ligand) fused to an Fc immunoglobulin domain, and optionally, the apoptosis inhibitor is APG101 or CAN008. [The present invention 1060] The method of claim 1022, claim 1048, or claim 1056, wherein the apoptosis inhibitor inhibits the activation or activity of a caspase, and optionally the caspase is caspase 2, caspase 8, caspase 9, caspase 10, caspase 3, caspase 6, or caspase 7, and optionally the caspase is caspase 3. [This invention 1061] 1060, wherein the apoptosis inhibitor is selected from the group consisting of emricasan (IDN-6556, PF-03491390), NAIP (neuronal inhibitor of apoptosis protein; BIRC1), cIAP1 and cIAP2 (cellular inhibitor of apoptosis 1 and 2; BIRC2 and BIRC3, respectively), XIAP (X-chromosome-linked IAP; BIRC4), survivin (BIRC5), BRUCE (Apollon; BIRC6), livin (BIRC7), and Ts-IAP (testis-specific IAP; BIRC8), wedelolactone, NS3694, NSCI, and Z-fluoromethylketone Z-VAD-FMK, or a fluoromethylketone variant thereof. [This invention 1062] The method of any of claims 1022, 1048, 1056, 1060, and 1061, wherein said apoptosis inhibitor is emericasan. [This invention 1063] The method of any one of claims 1022, 1048, 1056, and 1060 to 1062, wherein the apoptosis inhibitor is a pan-caspase inhibitor that inhibits the activation or activity of two or more caspases. [This invention 1064] Any of the methods of present inventions 1022, 1048, 1056, and 1060 to 1063, wherein the apoptosis inhibitor is Z-VAD-FMK, Z-FA-FMK, Z-VAD(OH)-FMK, Z-DEVD-FMK, Z-VAD(OM2)-FMK, or Z-VDVAD-FMK. [This invention 1065] The concentration of the apoptosis inhibitor is from 0.5 μM or about 0.5 μM to 50 μM or about 50 μM, 0.5 μM or about 0.5 μM to 25 μM or about 25 μM, 0.5 μM or about 0.5 μM to 10 μM or about 10 μM, 0.5 μM or about 0.5 μM to 5 μM or about 5 μM, 0.5 μM or about 0.5 μM to 1 μM or or about 1 μM, 1 μM or about 1 μM to 100 μM or about 100 μM, 1 μM or about 1 μM to 50 μM or about 50 μM, 1 μM or about 1 μM to 25 μM or about 25 μM, 1 μM or about 1 μM to 10 μM or about 10 μM, 1 μM or about 1 μM to 5 μM or about 5 μM, 5 μM or about 5 μM to 100 μM M or about 100 μM, 5 μM or about 5 μM to 50 μM or about 50 μM, 5 μM or about 5 μM to 25 μM or about 25 μM, 5 μM or about 5 μM to 10 μM or about 10 μM, 10 μM or about 10 μM to 100 μM or about 100 μM, 10 μM or about 10 μM to 50 μM or about 50 μM, 10 μM or is from about 10 μM to 25 μM or about 25 μM, from 25 μM or about 25 μM to 100 μM or about 100 μM, from 25 μM or about 25 μM to 50 μM or about 50 μM, or from 50 μM or about 50 μM to 100 μM or about 100 μM, inclusive. [The present invention 1066] The method of any one of claims 1025 to 1065, wherein said antigen-presenting cells are dendritic cells, mononuclear phagocytes, B lymphocytes, endothelial cells, or thymic epithelium. [This invention 1067] The method of any one of claims 1025 to 1066, wherein the antigen-presenting cells are dendritic cells. [The present invention 1068] The method of any of claims 1025 to 1067, wherein said antigen-presenting cells are autologous to said subject. [The present invention 1069] 1069. The method of any of claims 1025 to 1068, wherein said one or more non-natural peptides comprise individual peptides or a pool of peptides. [The present invention 1070] 1069. The method of any of claims 1025 to 1069, wherein the one or more non-natural peptides are loaded onto antigen-presenting cells by tandem transfection of in vitro transcribed synthetic minigene constructs encoding the one or more non-natural peptides, wherein the transcribed minigene constructs generate the individual peptides. [This invention 1071] 1070. The method of any of claims 1025 to 1070, wherein said one or more non-natural peptides are loaded onto antigen-presenting cells by peptide pulsing, optionally by electroporation. [This invention 1072] 1071. The method of claim 1071, wherein said one or more non-natural peptides are 5 to 30 amino acids in length, optionally 12 to 25 amino acids in length, optionally 25 amino acids in length or about 25 amino acids in length. [This invention 1073] the one or more non-natural peptides are a peptide pool, and the concentration of peptides in the peptide pool for the peptide pulse is from 0.001 μg / mL or about 0.001 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL to 40 μg / mL or about 40 μg / mL, 0.1 μg / mL or about 0.1 μg / mL to 40 μg / mL or about 40 μg / mL, 1 μg / mL or about 1 μg / mL to 40 μg / mL or about 40 μg / mL, 0.01 μg / mL or about 0.01 μg / mL to 10 μg / mL or about 10 μg / mL, or 1 μg / mL or about 1 μg / mL to 10 μg / mL or about 10 μg / mL; or The one or more non-natural peptides are individual peptides, and the concentration of the individual peptides for the peptide pulse is from 0.00001 μg / mL or about 0.00001 μg / mL to 1 μg / mL or about 1 μg / mL, from 0.00001 μg / mL or about 0.00001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, from 0.00001 μg / mL or about 0.00001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL. L, 0.0001 μg / mL or about 0.0001 μg / mL to 1 μg / mL or about 1 μg / mL, 0.0001 μg / mL or about 0.0001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, 0.0001 μg / mL or about 0.0001 μg / mL to 0.1 μg / mL or about 0.1 μg / mL, or 0.0001 μg / mL or about 0.0001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL, The method of invention 1071 or invention 1072. [This invention 1074] 1074. The method of any of claims 1071 to 1073, wherein the concentration of individual peptides of said one or more non-natural peptides is, on average, from 0.00001 μg / mL or about 0.00001 μg / mL to 0.01 μg / mL or about 0.01 μg / mL. [This invention 1075] 1075. The method of any of claims 1071 to 1074, wherein the concentration of individual peptides of said one or more non-natural peptides is, on average, from 0.0001 μg / mL or about 0.0001 μg / mL to 0.001 μg / mL or about 0.001 μg / mL. [This invention 1076] Any of the methods of inventions 1025 to 1075, wherein the co-culture ratio of antigen-presenting cells and T cells is 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 5:1 to 1:1, 2.5:1 to 1:1, 1:20 to 1:1, 1:15 to 1:1, 1:10 to 1:1, 1:5 to 1:1, or 1:2.5 to 1:1. [This invention 1077] 1076. The method of any of claims 1025 to 1076, wherein the co-culture ratio of dendritic cells to T cells is 5:1 to 1:5, or 3:1 to 1:3, and optionally 1:1 or about 1:1. [This invention 1078] The method of any of claims 1025 to 1077, wherein the co-culture ratio of antigen-presenting cells to T cells is 1:1 or about 1:1. [This invention 1079] 1079. The method of any one of claims 1025 to 1078, wherein the co-culturing step lasts for 2 hours to 24 hours. [The present invention 1080] 1079. The method of any of claims 1025 to 1079, wherein said co-culturing step lasts for 6 hours or about 6 hours. [This invention 1081] The method of any one of claims 1001 to 1080, wherein the step of selecting the cells is carried out using a fluorescence-based cell sorter. [This invention 1082] 1081. The method of claim 1081, wherein said fluorescence-based cell sorter is an automated high-throughput flow cytometry sorter, optionally an FX500 cell sorter or a Miltenyi Tyto cell sorter. [This invention 1083] 1083. The method of claim 1081 or 1082, wherein said selection is by one run, two runs, three runs, or four runs with a fluorescence-based cell sorter. [This invention 1084] 1084. The method of any of claims 1081 to 1083, wherein said selection is carried out using a fluorescence-based disposable fluidic cell sorter at a rate of 10,000 to 100,000 cells / second. [This invention 1085] 1023. The method of any one of claims 1001 to 1022, wherein the step of increasing is carried out in a closed system using a gas-permeable membrane. [The present invention 1086] The method of any one of claims 1001 to 1022, wherein the step of increasing is carried out in a closed system using a bioreactor. [This invention 1087] The method of any one of claims 1016 to 1084, wherein the first expansion step is carried out in a closed system using a gas-permeable culture vessel. [This invention 1088] The method of any one of claims 1016 to 1084, wherein the first expansion step is carried out in a closed system using a bioreactor. [This invention 1089] The method of any one of claims 1016 to 1088, wherein the step of carrying out the second expansion is carried out in a gas-permeable culture vessel. [The present invention 1090] The method of any one of claims 1016 to 1088, wherein the second expansion step is carried out using a bioreactor. [This invention 1091] The method of any one of claims 1001 to 1090, wherein the tumor is an epithelial cancer tumor. [This invention 1092] The method of any one of claims 1001 to 1091, wherein the tumor is a melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, gastric cancer, or uterine cancer tumor. [This invention 1093] The method of any one of claims 1001 to 1092, wherein the tumor is melanoma. [This invention 1094] The method of any of claims 1001 to 1093, wherein said tumor is colorectal cancer (CRC). [This invention 1095] Any of the methods of claims 1001 to 1094, wherein the tumor is a non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer tumor, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer. [This invention 1096] Any of the methods of inventions 1001 to 1095, resulting in a fold expansion of T cells or a fold expansion of tumor-reactive T cells from the input sample that is at least 2-fold or at least about 2-fold, at least 5-fold or at least about 5-fold, at least 10-fold or at least about 10-fold, at least 25-fold or at least about 25-fold, at least 50-fold or at least about 50-fold, at least 100-fold or at least about 100-fold, at least 250-fold or at least about 250-fold, at least 500-fold or at least about 500-fold, at least 750-fold or at least about 750-fold, at least 1000-fold or at least about 1000-fold, at least 1500-fold or at least about 1500-fold, at least 2000-fold or at least about 2000-fold, at least 2500-fold or at least about 2500-fold, or at least 3000-fold or at least about 3000-fold. [This invention 1097] 1096. The method of any of claims 1001 to 1096, wherein said composition of tumor-reactive cells produced by said method is capable of producing IFNγ at a concentration of greater than or greater than about 30 pg / mL, optionally greater than or greater than about 60 pg / mL, after antigen-specific stimulation. [This invention 1098] The method of any of claims 1001 to 1097, further comprising the step of harvesting the cells produced by said method for formulation as a therapeutic composition. [This invention 1099] 1098. The method of claim 1098, comprising formulating the harvested cells with a cryoprotectant. [The present invention 1100] A composition comprising tumor-reactive T cells produced by any of the methods of the present inventions 1001 to 1099. [The present invention 1101] The composition of claim 1100, comprising a cryoprotectant. [The present invention 1102] The composition of invention 1100 or invention 1101, wherein said T cells are CD3+ T cells or comprise CD4+ T cells and / or CD8+ T cells. [The present invention 1103] Any of the compositions of the present inventions 1100 to 1102, wherein the T cells comprise CD4+ T cells and CD8+ T cells, and the ratio of CD8+ T cells to CD4+ T cells is from 1:100 or about 1:100 to 100:1 or about 100:1, 1:50 or about 1:50 to 50:1 or about 50:1, 1:25 or about 1:25 to 25:1 or about 25:1, 1:10 or about 1:10 to 10:1 or about 10:1, 1:5 or about 1:5 to 5:1 or about 5:1, or 1:2.5 or about 1:2.5 to 2.5:1 or about 2.5:1. [The present invention 1104] the number of tumor-reactive T cells or the number of viable cells thereof in the composition is 0.5×10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 0.5×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 pieces, 0.5×10 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 pieces, 1×10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 1×10 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 1×10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 1×10 8 or approximately 1 x 108 From 60 x 10 8 or approximately 60 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 1×10 8 or approximately 1 x 10 8 From 3.5 x 10 8 or approximately 3.5 x 10 8 pieces, 3.5×10 8 or approximately 3.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 3.5×10 8 or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 3.5×10 8 or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 3.5×10 8 or approximately 3.5 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 3.5×10 8 or approximately 3.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 3.5×10 8 or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 8×10 8 pieces or approximately 8 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 15×10 8 pieces or approximately 15 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 pieces, 60×10 8 or approximately 60 x 10 8 From 50 x 109 or approximately 50 x 10 9 pieces, 60×10 8 or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, 60×10 8 or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 pieces, 12×10 9 pieces or approximately 12 x 10 9 From 30 x 10 9 pieces or approximately 30 x 10 9 pieces, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 or approximately 60 x 10 9 The composition of any one of 1100 to 1103 of the present invention, wherein the number of the compounds is 1 (including both end values). [This invention 1105] the number of tumor-reactive T cells or the number of viable cells thereof in the composition is at least 5×10 8 or at least about 5 x 10 8 The composition of any one of the present inventions 1100 to 1104. [The present invention 1106] the number of tumor-reactive T cells or the number of viable cells thereof in the composition is at least 1 x 10 9 or at least about 1 x 10 9 The composition of any one of the present inventions 1100 to 1104. [This invention 1107] the number of tumor-reactive T cells or the number of viable cells thereof in the composition is at least 10×10 9 or at least about 10 x 10 9 The composition of any one of the present inventions 1100 to 1104. [This invention 1108] The composition of any one of claims 1100 to 1107, comprising a pharmaceutically acceptable excipient. [This invention 1109] A therapeutic method comprising the step of administering any one of the compositions of the present inventions 1100 to 1108 to a subject having cancer. [The present invention 1110] 1109. The method of claim 1109, wherein said cells of said administered composition are autologous to said subject. [The present invention 1111] The therapeutically effective dose is 1 × 10 8 ~10×10 9 The method of claim 1109 or 1110, wherein the T cells are T cells or live cells thereof. [The present invention 1112] The therapeutically effective dose is 5 × 10 8 ~10×10 9 The method of claim 1109 or 1110, wherein the T cells are T cells or live cells thereof. [The present invention 1113] The therapeutically effective dose is 5 × 10 8 ~1×10 9 The method of claim 1109 or 1110, wherein the T cells are T cells or live cells thereof. [This invention 1114] The method of any one of claims 1109 to 1113, wherein the cancer is epithelial cancer. [This invention 1115] The method of any one of claims 1109 to 1114, wherein said cancer is melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer. [The present invention 1116] Any of the methods of claims 1109 to 1115, wherein the cancer is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer. [This invention 1117] 1109. Any of the compositions of claims 1100 to 1108 for use in treating a subject having cancer. [This invention 1118] Use of any of the compositions of inventions 1100 to 1108 for the manufacture of a medicament for treating a subject with cancer. [This invention 1119] 1117. A composition for use according to claim 1117 or a use according to claim 1118, wherein said cells of said composition to be administered are autologous to said subject. [The present invention 1120] The therapeutically effective dose is 1 × 10 8 ~10×10 9 A composition for use or use according to any one of claims 1117 to 1119, which is a T cell or a live cell thereof. [This invention 1121] The therapeutically effective dose is 5 × 10 8 ~10×10 9 A composition for use or use according to any one of claims 1117 to 1119, which is a T cell or a live cell thereof. [This invention 1122] The therapeutically effective dose is 5 × 10 8 ~1×10 9 A composition for use or use according to any one of claims 1117 to 1119, which is a T cell or a live cell thereof. [This invention 1123] The composition for use or use of any of claims 1117 to 1122, wherein the cancer is epithelial cancer. [This invention 1124] The composition or use of any of inventions 1117 to 1122, wherein the cancer is melanoma, lung squamous cell carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer. [This invention 1125] The composition or use of any of claims 1117 to 1124, wherein the cancer is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer. [Brief explanation of the drawings]

[0069] [Figure 1A]

[0023] Figures 1A and 1B show a schematic diagram of an exemplary process for producing a T cell therapy composition according to the provided methods. In the exemplary process, a tumor sample is obtained from a patient for identification and generation of peptides for use in co-culture with autologous T cells obtained from the same subject. In some cases, a T cell population obtained from the patient, containing, for example, tumor-infiltrating lymphocytes (TILs) or peripheral blood lymphocytes (PBLs), is stimulated under conditions for cell expansion before co-culture with antigen-presenting cells that have been contacted or exposed to peptide neoepitopes for presentation on the major histocompatibility complex. After co-culture under conditions for antigen-presenting cells to present the peptides in the context of the major histocompatibility complex, tumor-reactive T cells, or T cells surface-positive for one or more T cell activation markers associated with tumor-reactive T cells (e.g., CD39, PD-1, and / or TIGIT), can be selected and cultured under conditions for expansion according to the provided methods, such as incubation with a T cell stimulator (e.g., IL-2 and / or anti-CD3 / anti-CD28). In some embodiments of the provided methods, tumor-reactive T cells are selected directly from a tumor sample or a digested sample from a tumor sample that has been subjected to initial (e.g., minimal) expansion, and such methods do not include a co-culture step with antigen-presenting cells that present peptide neoepitopes. Steps may include incubation with T cell stimulators and / or other T cell adjuvants, according to the provided methods. The culturing step may be performed in the presence of one or more recombinant cytokines (e.g., IL-2) to support cell growth and expansion. This process may be performed in the presence of serum-free medium containing nutrients. One or more, or all, of the steps may be performed in a closed system, e.g., without exposing the cells to the environment. Once a therapeutic dose or threshold number of cells is reached, the cells are harvested and formulated, optionally concentrated or cryopreserved, and available for administration to a subject, e.g., by infusion. [Figure 1B]

[0023] Figure 1B shows a schematic diagram of an exemplary process for producing a T cell therapy composition according to the provided methods. In the exemplary process, a biological sample containing T cells is used as the cell source for the method. The biological sample may include tumor-infiltrating lymphocytes, peripheral blood mononuclear cells (e.g., apheresis), or lymphocyte-derived lymphocytes. Tumor-reactive T cells, or T cells surface-positive for one or more T cell activation markers associated with tumor-reactive T cells (e.g., CD39, PD-1, and / or TIGIT), can be selected directly from the sample and cultured under conditions for expansion according to the provided methods, including incubation with a T cell stimulator and / or a T cell adjuvant. The culturing step can be performed in the presence of one or more recombinant cytokines (e.g., IL-2) to support cell growth and expansion. The process can be performed in the presence of serum-free medium containing nutrients. One or more, or all, of the steps can be performed in a closed system, e.g., without exposing the cells to the environment. Once a therapeutic dose, or threshold number of cells, is reached, the cells can be harvested and formulated, optionally concentrated or cryopreserved, and available for administration to a subject, for example, by infusion. [Figure 1C]

[0023] Figure 1C shows a T cell manufacturing process according to certain embodiments described herein. Figure 1C shows a complete process flow chart for generating a patient-specific tumor-derived infiltrating T cell population. [Figure 2] Figure 2A shows exemplary kinetics and T cell neoantigen reactivity in a typical TIL expansion process, which includes bulk expansion of T cells with a first initial expansion and a second rapid expansion, where reactivity remains low throughout the process, including in the final product. Figure 2B further shows exemplary kinetics of the TIL expansion process provided herein, which includes a first initial expansion, followed by enrichment of tumor-reactive T cells by co-culture with neoantigen peptide-presenting antigen-presenting cells, selection of tumor-reactive cells for T cell activation (upregulation) markers, and a second expansion of the enriched reactive cells. [Figure 3] Figure 3A shows the generation of total viable population 1 cells from patient-derived CRC tumor tissue using fragment culture and enzymatic and non-enzymatic homogenization. Both enzymatic and non-enzymatic digestion yielded more total cells than cultures from fragments. The viability of these cells is shown in Figure 3B. The viability of enzymatically digested cultures generated from fragments was higher than that obtained using non-enzymatic homogenization. [Figure 4] Figure 4A shows the generation of Population 1 cells from patient-derived melanoma tumor tissue using fragment culture or homogenization with or without enzymes. Fragment cultures yielded more total cells than cultures initiated from single-cell suspensions. The viability of these cells is shown in Figure 4B. Populations generated from fragments exhibited higher viability than cells from single-cell suspensions. [Figure 5] Figure 5A shows the growth curves (Figure 5B) and fold expansion (Figure 5C) of Population 2 cells derived from primary CRC tumors in either conventional 6-well culture plates or 24-well gas-permeable culture plates. Figure 5 also shows the total cell number (Figure 5C) and fold expansion (Figure 5D) of Population 2 cells derived from primary CRC tumors compared by cell extraction method, either fragment culture or single-cell suspension culture. [Figure 6] Growth curves (FIG. 6A) and fold expansion (FIG. 6B) of Population 2 cells derived from primary melanoma tumors in either 6-well culture plates or 24-well gas-permeable culture plates are shown. [Figure 7] The total cell number (FIG. 7A) and fold expansion (FIG. 7B) of Population 2 cells derived from primary CRC tumors using serum-free OpTmizer medium or RPMI medium supplemented with 5% human serum are shown. [Figure 8] Similarly, Figure 8 shows the total cell number (Figure 8A) and fold expansion (Figure 8B) of Population 2 cells derived from primary melanoma tumors using serum-free OpTmizer medium or RPMI medium supplemented with 5% human serum. [Figure 9]Figure 9A shows the total cell number (Figure 9A) and fold expansion (Figure 9B) of Population 2 cells derived from CRC tumors and cultured in medium supplemented with either low (300 IU / mL) or high (6000 IU / mL) concentrations of recombinant human IL-2. These data are similarly shown for melanoma tumor-derived cells in Figures 10A-B. High concentrations of IL-2 were not observed to be required for cell expansion. [Figure 10] See legend to Figure 9. [Figure 11] Figure 11A shows the total cell counts of population 2, and Figure 11B shows that similar fold expansions were observed from melanoma-derived cell cultures that were unstimulated or stimulated with the anti-CD3 monoclonal antibody OKT3. [Figure 12] Figures 12A-C show the upregulation rates of activation markers on CD8+ T cells, CD38 and CD39 (Figure 12A), CD134 and CD137 (Figure 12B), and CD69 and CD90 (Figure 12C), from 0 to 48 hours after activation with OKT3. [Figure 13] Figures 13A-C show the upregulation rates of activation markers on CD4+ T cells, CD38 and CD39 (Figure 13A), CD134 and CD137 (Figure 13B), and CD69 and CD90 (Figure 13C), from 0 to 48 hours after activation with OKT3. [Figure 14] 1 shows the expression of selected exemplary markers in single cell suspension cultures generated from day 0 CRC tumors. [Figure 15]Figures 15A-E show CD3+ cell purity as a percentage of population 1 cells. Figure 15A shows the purity of cells derived from day 0 SCS from CRC tumors after homogenization without enzyme, homogenization with 1 mg / ml (low) enzyme, and homogenization with 5 mg / ml (high) enzyme. These data are similarly shown in Figure 15B for melanoma-derived cultures. Figure 15C shows the purity of CD3+ population 1 cells on day 0 (baseline SCS) and day 6 from fragments cultured with or without OKT3 stimulation. Figure 15D shows the relative purity of CD3+ cells from CRC donors on day 11 using fragments cultured in medium supplemented with either 6000 IU / mL (high) or 300 IU / mL (low) recombinant IL-2. Figure 15E shows population 1 cells (day 9) from fragments cultured in either serum-free OpTmizer medium or RPMI containing OKT3 stimulation and / or either high or low concentrations of IL-2. These observations support the idea that SCS derived from tumor biopsies of CRC patients may provide greater numbers of T cells for expansion than cells obtained from culture of tumor fragments. [Figure 16] 1 shows the purity of CD3+ Population 1 cells from melanoma patients as fragment cultures from day 9 using serum-containing RPMI medium or serum-free OpTmizer at high and low IL-2 concentrations. [Figure 17] Figure 17A shows the generation of Population 3 cells after co-culture with dendritic cells loaded with peptides at concentrations ranging from 0.1 ng / mL to 20 ng / mL. Figure 17B shows the fold increase in the same experiment from T cells co-cultured with non-loaded dendritic cells (Figure 17B). [Figure 18] Figure 18A compares stimulation with one or two peptides, reported as % 41BB / OX40 expression, and Figure 18B shows stimulation with one or two peptides, reported as fold increase from resting T cells. [Figure 19]Figure 19A compares two T cell to dendritic cell ratios of 1:1 and 1:2, reported as % 41BB / OX40 expression. Figure 19B compares two T cell to dendritic cell ratios of 1:1 and 1:2, reported as fold increase from resting T cells. [Figure 20] Figure 20A shows the neoantigen-reactive TCR rates before and after co-culture with autologous neoantigen peptides and sorting of T cells from peripheral blood of three healthy donors. Figure 20B shows the average class I reactivity of CD8+ cells before and after co-culture and sorting. [Figure 21] Figures 21A and 21B show recovery from cell sorting using the Sony FX500 as both total cell input and output for two independent runs (Figure 21A) and recovery percentages (Figure 21B). [Figure 22] Figure 1 shows the purity and gating of the CD4+ population from cell sorting using a Sony FX500. The results demonstrate the selection of cells positive for up-regulation markers and high recovery of cells after sorting. [Figure 23A] Figure 23 shows the expansion of tumor-infiltrating T lymphocytes after sorting. Figure 23A shows the total cell count. [Figure 23B] Figure 23B shows the expansion of tumor-infiltrating T lymphocytes after sorting. Figure 23B shows the fold expansion of population 5 cells from population 4 cells after co-culture in the presence or absence of dendritic cells loaded with wild-type peptide, tumor-associated peptide, or no peptide. [Figure 23C] Figure 23C shows the expansion of tumor-infiltrating T lymphocytes after sorting. The predicted cell numbers after expansion of population 4 cells into population 5 cells at various cell recovery numbers after sorting are shown in Figure 23C. [Figure 24A] Shown are measured IFN-γ secretion within bulk co-culture positively sorted (selected) populations by CD137 and / or CD134 expression from bulk co-cultured cells (enriched) or negatively sorted (unselected) populations from bulk co-cultured cells after stimulation with mutant (mut) or normal wild-type (WT) peptides obtained from ovarian cancer patients. [Figure 24B]1 shows enrichment of neo-antigen-specific populations of tumor-reactive specific cells in positive and negative sorting compared to bulk unsorted T cells. [Figure 24C] The number of TCR clonotypes present in the unselected and selected populations is shown, demonstrating a high diversity of incoming TCRs in the unselected T cell population and an enrichment for unique TCR clones in the selected population. [Figure 24D] Pre- and post-sort cell populations from sample A were observed to contain CD4+ and CD8+ cells, demonstrating that class I and class II reactive cells were present in the enriched population. [Figure 25A] Shown is measured IFN-γ secretion within a bulk co-culture positively sorted (selected) population by expression of CD137 and / or CD134 from bulk co-cultured cells (enriched) or a negatively sorted (unselected) population from bulk co-cultured cells after stimulation with anti-CD3 (OKT3) obtained from a colorectal cancer patient. [Figure 25B] 1 shows enrichment of neo-antigen-specific populations of tumor-reactive specific cells in positive and negative sorting compared to bulk unsorted T cells. [Figure 25C] 1 shows the TCR clonality profiles present in the unselected and selected populations. [Figure 25D] Pre- and post-sort cell populations observed to contain CD4+ and CD8+ cells are shown, demonstrating that class I and class II reactive cells are present in the enriched populations. [Figure 26A] Shown is the enrichment of neo-antigen-specific populations of tumor-reactive specific cells in bulk co-culture positively sorted (selected) populations by expression of CD137 and / or CD134 from bulk co-cultured cells (enriched) or in negatively sorted (unselected) populations from bulk co-cultured cells. [Figure 26B] 1 shows the TCR clonality profiles present in the unselected and selected populations. [Figure 26C]Pre-sort (bulk) and post-sort cell populations observed to contain both CD4+ class I-reactive and CD8+ class II-reactive cells are shown. [Figure 27] Figures 27A-C show total viable CD3+ cell counts for cells grown in the presence of multiple T cell adjuvants with supplemented OKT3 stimulation. Results shown are for the following adjuvants: 10 μg / mL of tabolixizumab, oxelumab, ipilimumab, tocilizumab, urelumab, pembrolizumab, varlilumab, anti-GITR MK-1248, anti-human FasL; 25 μM for the Z-VAD-FMK pan-caspase inhibitor; 250 nM for the HSP inhibitor NVP-HSP990; and 1000 IU / mL for cytokines (IL-7, IL-15, IL-21, IL-23, IL-25, IL-27, or IL-35). [Figure 28] Figures 28A-C show total viable CD3+ cell counts for cells grown in the presence of multiple T cell adjuvants without supplemental OKT3 stimulation. Results shown are for the following adjuvants: 10 μg / mL of tabolixizumab, oxelumab, ipilimumab, tocilizumab, urelumab, pembrolizumab, varlilumab, anti-GITR MK-1248, anti-human FasL; 25 μM for the Z-VAD-FMK pan-caspase inhibitor; 250 nM for the HSP inhibitor NVP-HSP990; and 1000 IU / mL for a cytokine (IL-7, IL-15, IL-21, IL-23, IL-25, IL-27, or IL-35). [Figure 29] Dose-response curves for IL-7 (Figure 29A) and IL-15 (Figure 29B) are shown. [Figure 30] Figures 30A-B through 32A-B show the total cell number and cell viability for cells derived from each of the three healthy donors and grown under the experimental conditions. Cells grown in the presence of sustained caspase inhibition were observed to exhibit superior growth despite inherent donor variability. [Figure 31] See legend to Figure 30. [Figure 32]See legend to Figure 30. [Figure 33] Figures 33A-B through 36A-B show the cell viability after sequential activation or transient activation with anti-CD3 / anti-CD28 (transient activation) for two donors. Figures 33A-B show the cell viability after single activation with anti-CD3 / anti-CD28 (transient activation) for two donors, and Figures 34A-B show the total cell counts for the same treatment. Figures 35A-B show the cell viability after sequential activation with anti-CD3 / anti-CD28 for two donors, and Figures 36A-B show the total cell counts for the same treatment. [Figure 34] See legend to Figure 33. [Figure 35] See legend to Figure 33. [Figure 36] See legend to Figure 33. [Figure 37] Figures 37A-C show the fold expansion (Figure 37A), total viable cells (Figure 37B), and viability (Figure 37C) of both SCS and tumor fragment-derived cultures grown in the presence or absence of the pan-caspase inhibitor Z-VAD-FMK. [Figure 38A] Figures 38A-D through 40A-D show the T cell phenotypes of T cells after incubation with various T cell adjuvants. T cell phenotypes are shown for CD3+ (Figures 38A-D), CD4+ (Figures 39A-D), and CD8+ (Figures 40A-D) cells grown in the presence of various concentrations of ipilimumab (anti-CTLA4), pembrolizumab (anti-PD1), taborixizumab (anti-TNFRSF4), urelumab (anti-CD137), and varlilumab (anti-CD27). [Figure 38B] See legend to Figure 38A. [Figure 38C] See legend to Figure 38A. [Figure 38D] See legend to Figure 38A. [Figure 39A] See legend to Figure 38A. [Figure 39B] See legend to Figure 38A. [Figure 39C] See legend to Figure 38A. [Figure 39D] See legend to Figure 38A. [Figure 40A] See legend to Figure 38A. [Figure 40B] See legend to Figure 38A. [Figure 40C] See legend to Figure 38A. [Figure 40D] See legend to Figure 38A. [Figure 41] Figures 41A-49A show total viable CD3+ cell counts for cells grown in the presence of IL-2 with additional regulatory cytokines or other T cell adjuvants. Results shown are for three concentrations of the following adjuvants: oxelumab (Figure 48A), anti-GITR MK-1248 (Figure 47A), and Z-VAD-FMK pan-caspase inhibitor (Figure 49A); and for cytokines: IL-23, IL-21, IL-35, IL-27, IL-15, and IL-7 (Figures 41A, 42A, 43A, 44A, 45A, and 46A). Figures 41B-49B show T cell phenotype as a function of naive and central memory cell populations in cells grown in the presence of three concentrations of oxelumab (Figure 48B), anti-GITR MK-1248 (Figure 47B), Z-VAD-FMK pan-caspase inhibitor (Figure 49B); and cytokines IL-23, IL-21, IL-35, IL-27, IL-15, IL-7 (Figures 41B, 42B, 43B, 44B, 45B, 46B). [Figure 42] See legend to Figure 41. [Figure 43] See legend to Figure 41. [Figure 44] See legend to Figure 41. [Figure 45] See legend to Figure 41. [Figure 46] See legend to Figure 41. [Figure 47] See legend to Figure 41. [Figure 48] See legend to Figure 41. [Figure 49] See legend to Figure 41. [Figure 50] Figures 50A-C show the CD4+ / CD8+ cell ratio assessed by flow cytometry at the end of the culture period after culture of cells from a representative healthy donor grown in the presence of IL-2 with additional regulatory cytokines or other T cell adjuvants. Neither the antibodies (Figure 50A), cytokines (Figure 50B), nor other regulatory factors (Figure 50C) tested substantially altered the CD4+ / CD8+ T cell ratio from that observed with IL-2 alone. DETAILED DESCRIPTION OF THE INVENTION

[0070] Detailed Description Methods for producing T cells are provided herein. Such methods include, but are not limited to, (1) selecting cells positive for chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells positive for exhaustion markers from among PD-1, CD39, and / or TIGIT from a cell population containing T lymphocytes obtained from a donor subject; and (2) stimulating the population by incubating or culturing the selected cells with one or more T cell stimuli for lymphocytes to generate an expanded T cell population. In some embodiments, the selection and / or stimulation methods are performed in a closed system. In some embodiments, only a single expansion step is performed in the method. In some embodiments, an initial expansion (e.g., first expansion) is performed before selecting the cells. In some embodiments, the provided methods can further include a secondary stimulation to further expand the cells, where the additional stimulation is by incubation or culture with one or more T cell stimuli. In some embodiments, after the first stimulation (first expansion) and before the second stimulation (second expansion), the method may further include (i) co-culturing the T cell population in the presence of antigen-presenting cells presenting one or more MHC-associated non-native peptides; (4) separating antigen-presenting cells from the T cell population in a closed system, such as by selecting T cells containing endogenous TCRs that are reactive to the peptides present on the APCs, e.g., based on upregulation or activation markers on the T cells after co-culture with the APCs / peptides. In some embodiments, one or more of the steps are performed in a closed system. In some embodiments, all of the steps are performed in a closed system.

[0071] According to the embodiments herein, a method or process is provided for producing a T cell preparation that can be useful for treating patients with pathological diseases or conditions.Compared to known production methods, the methods and processes described herein can be completed in a significantly shorter time and can recover a larger number of endogenous TCR-expressing T cells, thereby providing significant advantages for clinical use of cells at therapeutic doses.The T cell populations produced by the methods described herein and pharmaceutical compositions thereof are also provided herein.

[0072] The provided method relates to the generation of T cell therapies reactive against tumor-associated antigens, such as neoantigens. Cancer cells accumulate many different DNA mutations as part of the tumorigenesis process. These mutations can cause amino acid changes in protein-coding regions. For the mutations to be recognized by the immune system, the proteins must be processed intracellularly and presented on the surface by the major histocompatibility complex (MHC). Peptide neoantigens (also referred to herein as neoepitopes or peptide neoepitopes) are mutant peptides presented by the MHC complex that can be recognized by T cells via TCR binding. For the immune system to recognize the mutation, the mutation must be expressed on the surface of the cancer cell via the MHC complex, and the T cell must have a TCR that recognizes the mutant peptide. These neoantigens can be presented by MHC class I and MHC class II and are recognized by CD8+ T cells and CD4+ T cells, respectively.

[0073] In some embodiments, the described methods can be used to produce T cells expressing cell surface receptors. The cell surface receptor can be a T cell receptor (TCR) or a novel TCR family. In certain embodiments of the provided methods, the T cell population is or includes reactive T cells expressing cell surface receptors, such as T cell receptors (TCRs), that can recognize peptide antigens on the surface of target cells. Specifically, for antigens to be recognized by the immune system, the protein must be processed intracellularly into peptide fragments that are presented on the surface by major histocompatibility complex (MHC). TCRs have two protein chains designed to bind to specific peptides presented by major histocompatibility complex (MHC) proteins on the surface of specific cells. Because TCRs recognize peptides in the context of MHC molecules expressed on the surface of target cells, TCRs have the potential to recognize antigens not only directly presented on the surface of target cells, e.g., cancer cells, but also presented by antigen-presenting cells, e.g., in tumor microenvironments, inflammatory microenvironments, and infectious microenvironments, as well as in secondary lymphoid organs. Reactive T cells expressing such cell surface receptors can be used to target and kill any target cells, including, but not limited to, infected cells, damaged cells, or dysfunctional cells. Thus, according to the embodiments described herein, manufactured T cells expressing cell surface receptors can be used to target and kill any target cells, including, but not limited to, infected cells, damaged cells, or dysfunctional cells. Examples of such target cells can include cancer cells, virus-infected cells, bacteria-infected cells, dysfunctionally activated inflammatory cells (e.g., inflammatory endothelial cells), and cells involved in dysfunctional immune responses (e.g., cells involved in autoimmune diseases).

[0074] In some embodiments, a "T cell receptor" or "TCR" is a molecule that comprises a variable α chain and a variable β chain (also known as TCRα and TCRβ, respectively) or a variable γ chain and a variable δ chain (also known as TCRγ and TCRδ, respectively), or an antigen-binding portion thereof, and is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the αβ form. TCRs, which typically exist in the αβ and γδ forms, are generally structurally similar, although the T cells that express them may have different anatomical locations or functions. TCRs can be found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0075] In some aspects, the reactive T cells are tumor-reactive T cells that recognize cancer neoantigens. Most neoantigens arise from passenger mutations, which means they do not confer any growth advantage to cancer cells. A relatively small number of mutations actively promote tumor growth, and these are known as driver mutations. Passenger mutations are likely to generate neoantigens that are unique to each patient and may exist in every subset of cancer cells. Driver mutations generate neoantigens that are likely to exist in every tumor cell of an individual and potentially be shared. In some embodiments of the provided method, the T cell population contains tumor-reactive T cells that can recognize neoantigens that contain passenger mutations and / or driver mutations.

[0076] In certain aspects, the provided methods can be used for ex vivo generation of T cell therapy, including ex vivo expansion of autologous tumor-reactive T cells. In some aspects, neoantigens are ideal targets for immunotherapy because they are disease-specific targets. For example, such antigens are not commonly present in the body before cancer develops, are truly cancer-specific, are not expressed on normal cells, and are not subject to off-target immunotoxicity. Therefore, a patient's unique repertoire of neoantigens can elicit a potent immune response specific to cancer cells while avoiding normal cells. This is an advantage over other cell therapy targets that may not be disease-specific, as even low levels of target antigens on normal cells can result in severe, fatal autoimmune toxicity in the context of engineered therapies targeting common antigens. For example, in melanoma patients, an anti-MAGE-A3 TCR program was discontinued due to trial-related deaths caused by cross-reactivity with a similar target, MAGE-A12, which is expressed at low levels in the brain. A key challenge in cancer immunotherapy is the identification of cancer targets.

[0077] Recent clinical trials have demonstrated that T cells isolated from surgically resected tumors, bearing TCRs that recognize neoantigens, can expand the population of these neoantigen-reactive TILs, and reinfuse them into patients, potentially resulting in dramatic clinical benefit. This personalized therapy has resulted in remarkable clinical responses in selected patients with common epithelial tumors.

[0078] Existing methods for obtaining and generating tumor-reactive T cells are not entirely satisfactory. For example, directly isolating tumor-reactive T cells from a subject without expanding them is not feasible due to the inability to obtain therapeutically effective amounts of such cells. Alternatively, attempts have been made to identify TCRs specific to desired neoantigens for recombinant engineering of TCRs into T cells for use in adoptive cell therapy methods. However, such approaches only generate a single TCR against a particular neoantigen, thereby lacking the diversity to recognize a broader repertoire of multiple tumor-specific mutations. Other methods involve bulk expansion of T cells from a tumor source, which carries the risk of expanding T cells that are not reactive to tumor antigens and / or may include a large number of bystander cells that may exhibit inhibitory activity. For example, tumor regulatory T cells (Tregs) specialize in suppressing immune responses and can limit the reactivity of T cell products by targeting CD4 T cells. + These additional approaches to expand tumor-reactive T cells ex vivo are not selective, and as a result, non-reactive T cells in the culture may be preferentially expanded over reactive T cells, resulting in an end product lacking satisfactory reactivity and / or with insufficient numbers of tumor-reactive T cells. Methods for generating tumor-reactive T cells for therapy are needed.

[0079]

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

[0080] The provided methods result in enriched T cell populations that are reactive to the patient-specific mutation, e.g., based on selection of upregulated markers after presentation of the mutant antigen and / or based on expansion of T cells enriched for tumor-reactive T cells after co-culture with antigen-presenting cells presenting the peptide neoepitope. For example, methods of culturing cells include methods for growing and expanding cells, and in particular, enriching for the expansion and expansion of tumor-reactive T cells, e.g., by selection of such cells or based on specific selection markers associated with or indicative of tumor-reactive T cells.

[0081] In the provided methods, exemplary markers for selecting or enriching tumor-reactive T cells include CXCL13 and / or one or more exhaustion markers, e.g., one or more of PD-1, CD39, and TIGIT.

[0082] Chemokine (C-X-C motif) ligand 13 (CXCL13), also known as B lymphocyte chemoattractant (BLC) or B cell-attracting chemokine 1 (BCA-1), is a protein ligand encoded by the CXCL13 gene in humans. CXCL13 is a small chemokine belonging to the C-X-C chemokine family. As its name suggests, this chemokine is selectively chemotactic for B cells belonging to both the B-1 and B-2 subsets, and it exerts its effects by interacting with the chemokine receptor CXCR5. CXCL13 and its receptor CXCR5 regulate the organization of B cells within follicles in lymphoid tissues and are highly expressed in the human liver, spleen, lymph nodes, and intestine. The CXCL13 gene is located on human chromosome 4 within a cluster of other C-X-C chemokines. In T lymphocytes, CXCL13 expression is thought to reflect the germinal center origin of T cells, particularly a subset of T cells called follicular helper T cells (or T cells). Thus, in T cell lymphomas such as angioimmunoblastic T cell lymphoma, CXCL13 expression may reflect the germinal center origin of neoplastic T cells.

[0083] Evidence also indicates that the action of CXCL13 and its receptor CXCR5 through the CXCL13:CXCR5 axis modulates cell-cell interactions that regulate lymphocyte infiltration within the tumor microenvironment, thereby determining responsiveness to cytotoxic and immune-targeted therapies. One study characterized a subset of tumor-infiltrating lymphocytes (TILs) from non-small cell lung cancer with enhanced tumor recognition capabilities, possessing high PD-1 expression and constitutive CXCL13 secretion, which may mediate immune cell recruitment to tertiary lymphoid structures (Thommen et al., abstract In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia, PA: AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B050; Thommen et al. (2018) Nature Medicine, 24:994-1004). CXCL13 can be expressed and produced by specific TIL subpopulations, such as the TGFβ-dependent CD103+CD8+ subpopulation (Workel et al. (2019) Cancer Immunology Research, 7(5):784-796).

[0084] PD-1, CD39, and TIGIT are checkpoint molecules that can also be markers for exhausted T cells. They are also activation or upregulation markers in that their expression increases during tumor reactivity, a natural mechanism of immunosuppression of the host immune response. For example, the immune system is designed to shut itself down to avoid an overactive immune response and prevent inflammatory and autoimmune responses. Thus, an immune response initially develops against cancer, which can be prevented by upregulating specific checkpoint molecules, such as PD-1, CD39, and TIGIT, that can inhibit the immune response. Because these markers are upregulated on cells in which an immune response is developing, the provided methods contemplate that such markers serve as powerful markers for specifically enriching tumor-reactive T cells that express TCRs against tumor antigens or neoepitopes. By specifically selecting tumor-reactive cells based on these activation markers, the provided methods avoid the bulk expansion of T cells from tumor sources, which contain a large number of bystander cells, such as Tregs, that are not tumor-reactive or may exhibit inhibitory activity.

[0085] The provided methods are intended to result in improved TIL therapy enriched for tumor-reactive T cells that have a high potential for therapeutic efficacy against neoantigens for treating specific cancers by selecting cells during one or more steps of the ex vivo process for producing tumor-reactive T cells based on secreted CXCL13 and / or expression of one or more exhaustion markers, PD-1, CD39, and / or TIGIT. The provided methods result in a product containing tumor-reactive T cells that can target multiple mutations and / or contain hundreds of TCRs reactive to various tumor antigens. Thus, such tumor-reactive T cells offer advantages over existing methods in which cells are transduced to express a single neoepitope-reactive TCR.

[0086] In some embodiments, CXCL13 secretion and / or expression of one or more of PD-1, CD39, and / or TIGIT are used to enrich TILs immediately after tumor dissociation, at the end of tumor fragment culture or immediately after mechanical / enzymatic generation of a single cell suspension from the tumor fragment. In some embodiments, CXCL13-producing cells are isolated from either tumor fragment culture or a single cell suspension generated by enzymatic digestion. In some embodiments, PD-1, CD39, and / or TIGIT-expressing cells are isolated from either tumor fragment culture or a single cell suspension generated by enzymatic digestion. In some aspects, TILs are selected based on CXCL13 production in combination with PD1, TIGIT, and CD39 enrichment or any combination thereof. In aspects of the provided methods, after selection, the selected cells (e.g., cells positive for CXCL13 secretion or cells surface positive for one or more of PD1, TIGIT, and CD39, e.g., PD1+ / CD39+ / TIGIT+) can be expanded in the presence of one or more T cell stimulators. In some embodiments, the T cell stimulator can include any one or more recombinant cytokines IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, or IL-25, e.g., generally at least IL-2 or IL-15. In some embodiments, the T cell stimulator can further include an anti-CD3 antibody (e.g., OKT3). In some embodiments, the T cell stimulator includes an anti-CD3 antibody (OKT3) and / or a recombinant cytokine such as IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, etc. In some embodiments, stimulation of the cells or any culturing or incubation can be further performed with an apoptosis inhibitor, such as a Fas decoy or a caspase inhibitor, or any combination thereof.

[0087] In some embodiments of the provided methods, a source of potential tumor peptides is used to identify TCRs reactive to neoantigens in a process that includes expanding T cells reactive to tumor neoantigen peptides. The provided methods include ex vivo co-culture methods in which a T cell population from T cells present in or expanded from a biological sample (e.g., tumor fragments or peripheral blood or other T cell source) is contacted with or incubated in the presence of antigen-presenting cells that present neoantigen peptides. In certain aspects, the T cells and antigen-presenting cells are autologous to the tumor-bearing subject from whom the peptides were identified. The provided methods further include separating, enriching, and / or selecting tumor-reactive T cells from the co-culture prior to or in conjunction with additional ex vivo expansion.

[0088] In some embodiments, the expanded, unenriched TILs can be co-cultured with antigen-presenting cells (APCs), such as autologous dendritic cells pulsed with a pool of neoantigen (mutant) peptides identified by whole-exome sequencing. Activated neoantigen-specific TILs can then be enriched based on CXCL13 secretion and / or expression of one or more of PD-1, CD39, and / or TIGIT. In some aspects, after co-culture with APCs presenting neoantigen (mutant) peptides, tumor-reactive cell populations can be selected or enriched based on CXCL13 production. In some aspects, after co-culture with APCs presenting neoantigen (mutant) peptides, tumor-reactive cell populations can be selected or enriched based on surface upregulation or expression of PD-1, TIGIT, and / or CD39. In some aspects, after co-culture with APCs presenting neoantigen (mutant) peptides, tumor-reactive cell populations can be selected based on CXCL13 production in combination with PD1, TIGIT, and CD39 enrichment, or any combination thereof. In aspects of the provided methods, selected and enriched neoantigen-reactive TILs (e.g., cells positive for CXCL13 secretion or cells surface positive for one or more of PD1, TIGIT, and CD39, e.g., PD1+ / CD39+ / TIGIT+) can be expanded in the presence of one or more T cell stimulators, e.g., anti-CD3 antibody (OKT3), and or recombinant cytokines, e.g., IL-2, IL-7, IL-15, IL-21, IL-25, IL-23. In some embodiments, stimulation of the cells or any culturing or incubation can be further performed with an apoptosis inhibitor, e.g., Fas decoy or a caspase inhibitor, or any combination thereof.

[0089] Additionally, the provided methods include reducing or limiting the presence of bystander cells in the resulting product and / or enriching tumor-reactive T cells. In certain aspects, the use of regulatory cytokines, such as one or more of recombinant IL-23, recombinant IL-25, recombinant IL-27, or recombinant IL-35, and / or immunosuppressive blockers (e.g., against TGFβ or IDO), can help promote T cell functionality while destroying or reducing the activity of undesirable cells, such as suppressive Treg cells. In some aspects, such regulatory cytokines and / or immunosuppressive blockers can be particularly advantageous during the isolation of TILs from tumors as a result of suppressive factors within the tumor microenvironment. In some aspects, the provided use of such regulatory cytokines and / or immunosuppressive blockers can also be included during the expansion of tumor-reactive T cells after isolation or enrichment and coculture with APC / peptide neoepitopes. For example, in some embodiments, regulatory cytokines, e.g., one or more of recombinant IL-23, recombinant IL-25, recombinant IL-27, and / or recombinant IL-35, and / or immunosuppressive blockers (e.g., against TGFβ or IDO1), may prove beneficial in tumor cultures during the initial stimulation and expansion of TILs and the expansion of isolated or enriched neoantigen tumor-reactive T cells. In other examples, regulatory cytokines, e.g., one or more of recombinant IL-23, recombinant IL-25, recombinant IL-27, or IL-35, and / or immunosuppressive blockers (e.g., against TGFβ or IDO1), may prove beneficial in preventing immunosuppression of neoantigen tumor-reactive T cells during the initial stimulation and expansion of TILs from the suppressive tumor microenvironment and during expansion with stimulators (such as IL-2). In a further example, the presence of such regulatory cytokines and / or immunosuppressive blockers can optimize TIL recovery during initial stimulation and expansion in tumor cell cultures.

[0090] Figure 1A shows a schematic diagram of an exemplary process for producing a T cell therapy composition according to the provided methods. In the exemplary process, a tumor sample is obtained from a patient for identification and generation of peptides for use in co-culture with antigen-presenting cells (APCs) that present the peptides and with autoantigen-associated T cells obtained from the same subject. In some cases, TILs are enriched from the sample by selecting cells positive for one or more markers associated with tumor-reactive cells (hereinafter "selection markers"), e.g., CXCL13 and / or exhaustion markers such as PD-1 / CD39 / TIGIT. In some cases, a T cell population obtained from the patient, e.g., containing or enriched for tumor-infiltrating lymphocytes (TILs), is stimulated under conditions to expand the cells before co-culture with antigen-presenting cells that have been contacted or exposed to peptide neoepitopes for presentation on the major histocompatibility complex. After co-culture under conditions in which the antigen-presenting cells present the peptide in the context of a major histocompatibility complex, tumor-reactive T cells, or T cells positive for one or more markers associated with tumor-reactive cells (hereinafter "selection markers"), such as CXCL13, exhaustion markers, such as PD-1 / CD39 / TIGIT, and / or T cell activation markers (e.g., CD137 and / or CD134), can be selected and cultured under conditions for expansion according to the provided methods, such as incubation with a T cell stimulant (e.g., recombinant IL-2, anti-CD3). The culturing step can be performed in the presence of one or more recombinant cytokines (e.g., IL-2) to support cell growth and expansion. This process can be performed in the presence of serum-free medium containing nutrients. One or more or all of the steps can be performed in a closed system, e.g., without exposing the cells to the environment. Once a therapeutic dose or a threshold number of cells is reached, the cells can be harvested and formulated, optionally concentrated or cryopreserved, and used for administration to a subject, e.g., by infusion. Figure 1B shows an exemplary process in which a cryopreservation step can be performed after one or more of the steps.

[0091] In some described embodiments, the provided methods are performed without a co-culture step involving the incubation of antigen-presenting cells (APCs) that present tumor peptides and autoantigen T cells obtained from the same subject.

[0092] The provided methods offer advantages over existing methods for generating and expanding TILs because they include a step of enriching tumor-reactive cells, for example, by selecting T cells that are likely or suspected to be enriched in tumor-reactive T cells. In some cases, the methods can further enrich tumor-reactive T cells by co-culturing with peptide-presenting APCs, followed by selection of reactive T cell clones that upregulate one or more selection markers associated with such cells. This process enriches the initial small population of tumor-reactive T cells expanded from a biological sample (e.g., tumor) for cells that are tumor-reactive or likely to be tumor-reactive cells before a subsequent second expansion step, thereby facilitating the preservation and expansion of the cells of interest and limiting the expansion of bystander T cells, which may include cells that are not reactive to tumor antigens and / or exhibit inhibitory activity (Figure 2A). This is in contrast to existing methods that involve passive expansion of bulk T cells, in which all tumor-derived T cells are subjected to a first initial expansion, for example, using a high IL-2 concentration, followed by a second rapid expansion of T cells present after the initial expansion. In such other methods, while these alternative processes can significantly expand total viable cells (TVC), there is no step that actively ensures that tumor-reactive T cells are primarily expanded (Figure 2B). Furthermore, the provided methods are performed to maximize the number of tumor-reactive cells that can be harvested, for example, by co-culturing all of the cells expanded after a first expansion with peptide-presenting APCs, and then selecting cells positive for one or more activation markers from the total bulk cells after co-culture before a subsequent second expansion. In aspects of the provided methods, all steps of the method are performed in a closed system.

[0093] The provided methods include one or more features that provide or relate to an improved, more efficient, and / or more robust process for generating tumor-reactive T cell therapeutic compositions ex vivo. In particular, the present disclosure relates to methods that offer advantages over available methods for generating TIL therapeutic cell compositions. Such advantages include, for example, reduced cost, streamlining, improved enrichment of tumor-reactive T cells in the therapeutic composition, and increased efficacy of the therapeutic composition across, for example, a variety of subjects and tumor conditions.

[0094] In an aspect of the provided methods, it has been observed that expansion can be successfully performed using relatively low concentrations of recombinant IL-2 during one or both expansion stages. Many existing methods use IL-2 at concentrations as high as 6000 IU / mL for T cell expansion of TILs. However, high IL-2 concentrations can increase the cost of the process and can be limiting. In some cases, high IL-2 concentrations can have a negative impact on T cell differentiation by promoting effector T cell differentiation over early memory T cells, which may be more desirable in therapeutic T cell compositions. The provided methods can be performed using concentrations several times lower than 6000 IU / mL, e.g., concentrations less than or about 1000 IU / mL, e.g., from 300 IU / mL or about 300 IU / mL to 1000 IU / mL or about 1000 IU / mL. In certain embodiments, the concentration of IL-2 is 300 IU / mL or about 300 IU / mL.

[0095] In embodiments of the provided methods, the T cell population is obtained from a biological sample known to contain T cells. In some embodiments, the T cell population is enriched from a biological sample from a subject, particularly a human subject. The biological sample can be any sample containing a bulk population of T cells. In some embodiments, the biological sample is or comprises peripheral blood mononuclear cells. In some embodiments, the biological sample is a peripheral blood sample or a serum sample. In some embodiments, the biological sample is a lymph node sample. In some embodiments, the biological sample is a tumor sample. In some aspects, the bulk T cells can include tumor-infiltrating T cells (TILs). In some embodiments, the subject is a human subject. In some embodiments, the subject has cancer, a viral infection, a bacterial infection, or an inflammatory condition. In certain embodiments, the subject has cancer.

[0096] In aspects of the provided methods, the starting source of cells (input sample) in the method can be tumor fragments (e.g., fragments 1-8 mm in diameter) or a single-cell suspension preparation obtained from enzymatic digestion of tumor fragments. 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 selected depending on the tumor type or cancer, for example, to optimize or increase the expansion and enrichment of tumor-reactive T cells from the tumor. In one example, the cancer is melanoma, and the starting population of lymphocytes is, for example, tumor fragments from a resected tumor. In another example, the cancer is colorectal cancer, and the starting population of lymphocytes is a single-cell suspension obtained by enzymatic digestion of tumor fragments, for example, with collagenase.

[0097] In some embodiments, the method includes first co-culturing the expanded T cells with peptide-loaded autologous antigen-presenting cells. In some aspects of the method, a relatively low concentration of peptide or peptide pool (including multiple peptides, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more, or any value between any of the above), such as when each individual peptide is less than 20 ng / mL, or even as low as 0.1 ng / mL, can result in increased activation of T cells in culture. In some embodiments, this can result in improved enrichment of tumor-reactive T cells in the co-culture before enriching cells by selection therefrom. In some embodiments, the co-culturing step in the provided methods comprises loading the APCs with individual peptides, or peptide pools, at a ratio of tumor-derived cells containing T cells to autologous APCs (e.g., dendritic cells) of at or about 1:5, to at or about 5:1, e.g., 1:3, to at or about 3:1, e.g., 1:1 or about 1:1. In some embodiments, the APCs are loaded with peptides or peptide pools at a concentration such that the individual peptides, or individual peptides of the peptide pool, are on average less than or about 20 ng / mL, e.g., from at or about 0.1 ng / mL to at or about 1 ng / mL, e.g., 0.1 ng / mL or about 0.1 ng / mL.

[0098] In some embodiments, the provided methods include enriching T cells, e.g., CD3+ T cells, or CD4 and / or CD8 subsets thereof, further based on one or more markers whose expression is upregulated on or specific to reactive or activated T cells (e.g., compared to resting or non-activated T cells) (hereinafter "reactive T cell markers" or T cell activation markers). Reactive T cells express a particular reactive marker when their endogenous TCR recognizes an antigen on a target cell or target tissue, e.g., when the TCR recognizes a neoantigen on a tumor. Exemplary reactive T cell markers include one or more, e.g., two, three, four, or more, of CD107, CD107a, CD39, CD103, CD137(4-1BB), CD59, CD90, CD38, CD30, CD154, CD252, CD134(OX40), CD258, CD256, PD-1, TIM-3, or LAG-3. Enrichment or selection of cells positive for one or more such upregulated markers on reactive or activated T cells can be performed before or during one or more steps of the expansion method. In certain embodiments, the provided methods involve enrichment or selection of cells positive for one or more upregulated markers on reactive or activated T cells after activation of the T cell population by coculture incubation with peptide-presenting APCs (e.g., DCs). In some embodiments, selecting cells positive for one or more upregulated markers on reactive or activated T cells from the co-culture can result in a two-fold or greater enrichment of antigen-specific tumor-reactive T cells and / or a substantial reduction in TCR clonality, demonstrating enrichment of TCR clonotypes consistent with enrichment of tumor-reactive T cells. Furthermore, such enriched T cells can exhibit improved ability to produce IFN-γ following antigen-specific stimulation compared to unselected or bulk T cells from the co-culture.

[0099] In some embodiments, the methods generate or expand T cells for use in adoptive cell therapy to treat diseases or conditions in which cells or tissues associated with the disease or condition are known or suspected to express an antigenic target recognized by the T cells. In some embodiments, the T cell therapy is autologous to the subject. In some embodiments, the T cell therapy is allogeneic to the subject.

[0100] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0101] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0102] I. Ex vivo expansion of tumor-reactive T cells The provided methods include ex vivo expansion and generation of T cell therapy compositions, particularly for use in connection with the treatment of cancer. In some embodiments, the manufacturing methods involve the propagation and manipulation of patient cells outside the body. In particular embodiments, the methods relate to methods for expanding T cells containing an endogenous TCR specific for 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 tumor antigens or T cells that are likely or suspected to be tumor-reactive T cells due to upregulation or positive surface expression of proteins expressed on T cells, such as T cell activation markers, that are expressed only when the T cell's endogenous TCR recognizes a peptide expressed by an APC. In some aspects, the frequency of these cells may be low, and ex vivo methods of enrichment and expansion are required to expand these cells to therapeutic doses.

[0103]

[0003] Provided embodiments relate to a process for preparing a therapeutic TIL composition enriched in tumor-reactive T cells, the process comprising direct selection of cells to obtain a further expanded tumor-reactive cell population. In the provided methods, a TIL tumor sample containing or predicted to contain tumor-reactive T cells (e.g., a first population) is obtained. In one embodiment, this population can be processed by digestion to produce a single-cell suspension (e.g., a second population). In some embodiments, this second population can be sorted to select cells enriched in tumor-reactive T cells (e.g., based on selection of cells positive for PD-1, CD39, and / or TIGIT) to obtain a selected or sorted cell population (e.g., a third population), which can then be expanded to produce a therapeutic composition containing an expanded tumor-specific reactive cell population (e.g., a fourth population). In other embodiments, the first population is processed or digested into fragments to create a single cell suspension (e.g., a second population), and then this second population is first subjected to initial expansion (e.g., a minimum expansion of 1-14 days) to obtain an initial cell population (e.g., a third population) that is sorted / selected for tumor-reactive T cells. In such embodiments, this third population is sorted to select cells enriched for tumor-reactive T cells (e.g., based on selection of cells positive for PD-1, CD39, and / or TIGIT) to obtain a selected or sorted cell population (e.g., a fourth population), which can then be expanded to produce a therapeutic composition containing an expanded tumor-specific reactive cell population (e.g., a fifth population).

[0104] In some embodiments, sorting and selection are performed for tumor-upregulated activation markers CD39, PD1, or TIGIT, or any combination thereof. Tumors containing tumor-recognizing reactive T cells upregulate activation markers such as CD39, PD1, and TIGIT. In the provided methods, T cells are sorted immediately after tumor digestion or after a short period of cell culture for cells surface-positive for CD39, PD1, and TIGIT to generate a tumor neoantigen-reactive T cell population. This process removes non-reactive and inhibitory "bystander" cells, resulting in a T cell product enriched for neoantigen-reactive T cells. The cells are then expanded to produce a clinically relevant number of tumor-specific T cells. In some embodiments, the final expanded therapeutic composition is formulated with a cryoprotectant for cryopreservation.

[0105] In one aspect of the provided methods, tumor-reactive T cells are selected directly after tumor digestion and a single expansion step is performed, in which case the expanded T cell population is collected as a therapeutic TIL composition. In such an example, tumor fragments are digested into a single cell suspension and provided as an input sample for sorting / selecting the tumor-reactive T cells. The selected cells are then expanded and collected as a therapeutic TIL composition. In some embodiments, expansion is performed over a period of time to achieve a therapeutic dose. In some embodiments, expansion is performed to achieve a cell expansion factor of at or about 200-fold to at or about 3000-fold. In some embodiments, expansion is performed to achieve a therapeutic dose of at or about 500 million or more than at or about 500 million total cells. In some embodiments, the expansion occurs over a period of 1 to 28 days, e.g., 7 to 28 days, 7 to 21 days, 7 to 14 days, or about 7 to 28 days, 7 to 21 days, 7 to 14 days, e.g., 7 days, 8 days, 9 days, 10 days, 11 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0106] In some embodiments, provided herein are methods for producing tumor-reactive T cells, comprising: (a) selecting, from an input sample containing T cells from a subject with a tumor, cells secreting chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells surface-positive for C-X-C chemokine receptor type 5 (CXCR5) to obtain selected cells from the sample; and (b) expanding the selected cells by culturing them with one or more T cell stimulants for lymphocytes under conditions to generate an expanded T cell population. In some embodiments, the method includes harvesting the expanded T cell population generated by the method for formulation as a therapeutic composition.

[0107] In some embodiments, provided herein are methods for producing tumor-reactive T cells, comprising: (a) selecting, from an input sample comprising T cells from a subject having a tumor, cells surface-positive for PD-1, CD39, and / or TIGIT to obtain selected cells from the sample; and (b) expanding the selected cells by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to generate an expanded T cell population. In some embodiments, the method comprises harvesting the expanded T cell population generated by the method for formulation as a therapeutic composition.

[0108] In some embodiments, provided herein are methods for producing tumor-reactive T cells, comprising: (a) selecting, from an input sample comprising T cells from a subject with a tumor, cells that are surface positive for PD-1 and CD39 to obtain selected cells from the sample; and (b) expanding the selected cells by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to produce an expanded T cell population. In some embodiments, the method comprises harvesting the expanded T cell population produced by the method for formulation as a therapeutic composition.

[0109] In some embodiments, provided herein are methods for producing tumor-reactive T cells, comprising: (a) selecting, from an input sample comprising T cells from a subject with a tumor, cells that are surface-positive for PD-1 and TIGIT to obtain selected cells from the sample; and (b) expanding the selected cells by culturing the selected cells with one or more T cell stimulants for lymphocytes under conditions to generate an expanded T cell population. In some embodiments, the method comprises harvesting the expanded T cell population generated by the method for formulation as a therapeutic composition.

[0110] In some embodiments, provided herein are methods for producing tumor-reactive T cells, comprising: (a) selecting surface-positive cells for CD39 and TIGIT from an input sample containing T cells from a subject with a tumor to obtain selected cells from the sample; and (b) expanding the selected cells by culturing the selected cells with one or more T cell stimulants for lymphocytes under conditions to produce an expanded T cell population. In some embodiments, the method comprises harvesting the expanded T cell population produced by the method for formulation as a therapeutic composition.

[0111] In some embodiments, provided herein are methods for producing tumor-reactive T cells, comprising: (a) selecting cells surface-positive for PD-1, CD39, and TIGIT from an input sample containing T cells from a subject with a tumor to obtain selected cells from the sample; and (b) expanding the selected cells by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to produce an expanded T cell population. In some embodiments, the method comprises harvesting the expanded T cell population produced by the method for formulation as a therapeutic composition.

[0112] In another aspect of the provided methods, at least two separate expansion steps are performed, where both expansions are performed after selecting or sorting cells for tumor-reactive T cells (e.g., for cells surface-positive for PD-1, CD39, and / or TIGIT). Thus, the incubation for expansion and harvesting is divided into two expansions. For example, the provided methods include a step of selecting cells surface-positive for a marker of tumor-reactive T cells (e.g., cells surface-positive for PD-1, CD39, and / or TIGIT), followed by a first expansion of the selected cells to obtain a first expanded cell population, and an additional (second) expansion of the first expanded cell population to obtain a second expanded cell population. In such examples, the second expanded T cell population is harvested as a therapeutic TIL composition. In some embodiments, the first and second expansions are performed together over a period of time to achieve a therapeutic dose. In some embodiments, the first expansion is performed to achieve a cell expansion factor of 2-fold or about 2-fold to 20-fold or about 20-fold. In some embodiments, the first expansion is carried out over a period of 1 to 14 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or less. In some embodiments, the second expansion is carried out to achieve an expansion factor of cells of at or about 100-fold to at or about 3000-fold. In some embodiments, the second expansion is carried out over a period of 7 or about 7 days to 21 days, e.g., about 14 days. In some embodiments, the first and second expansions are performed to achieve a therapeutic dose of 500 million, or about 500 million, or more than 500 million, or more than about 500 million total cells.

[0113] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) selecting cells surface-positive for PD-1, CD39, and / or TIGIT from an input sample containing T cells from a subject with a tumor to obtain selected cells from the sample; (b) performing a first expansion by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; and (c) performing a second expansion by culturing the first expanded cell population with one or more T cell stimuli of lymphocytes under conditions to generate a second expanded T cell population. In some embodiments, the methods comprise harvesting the second expanded T cell population produced by the methods for formulation as a therapeutic composition.

[0114] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) selecting cells surface-positive for PD-1 and CD39 from an input sample containing T cells from a subject with a tumor to obtain selected cells from the sample; (b) performing a first expansion by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; and (c) performing a second expansion by culturing the first expanded cell population with one or more T cell stimuli of lymphocytes under conditions to generate a second expanded T cell population. In some embodiments, the methods comprise harvesting the second expanded T cell population produced by the methods for formulation as a therapeutic composition.

[0115] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) selecting cells surface-positive for PD-1 and TIGIT from an input sample containing T cells from a subject with a tumor to obtain selected cells from the sample; (b) performing a first expansion by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; and (c) performing a second expansion by culturing the first expanded cell population with one or more T cell stimuli of lymphocytes under conditions to generate a second expanded T cell population. In some embodiments, the methods comprise harvesting the second expanded T cell population generated by the methods for formulation as a therapeutic composition.

[0116] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) selecting cells surface-positive for CD39 and TIGIT from an input sample containing T cells from a subject with a tumor to obtain selected cells from the sample; (b) performing a first expansion by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; and (c) performing a second expansion by culturing the first expanded cell population with one or more T cell stimuli of lymphocytes under conditions to generate a second expanded T cell population. In some embodiments, the methods comprise harvesting the second expanded T cell population produced by the methods for formulation as a therapeutic composition.

[0117] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) selecting cells surface-positive for PD-1, CD39, and TIGIT from an input sample containing T cells from a subject with a tumor to obtain selected cells from the sample; (b) performing a first expansion by culturing the selected cells with one or more T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; and (c) performing a second expansion by culturing the first expanded cell population with one or more T cell stimuli of lymphocytes under conditions to generate a second expanded T cell population. In some embodiments, the methods comprise harvesting the second expanded T cell population produced by the methods for formulation as a therapeutic composition.

[0118] In another aspect of the provided methods, the first expansion is performed before selecting or sorting the cells for tumor-reactive markers. In some embodiments, the first expansion is a minimal expansion performed under conditions and for a period of time such that activation markers (e.g., PD-1, CD39, and / or TIGIT) that are upregulated on cells of the collected tumor sample are still present during the sorting step and are not downregulated during the first expansion. In some embodiments, the initial expansion is carried out for 1 to 14 days, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, or for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or less. In such embodiments, the method includes sorting or selecting cells from the initially expanded T cell population. After sorting and selection of tumor-reactive T cells (e.g., cells surface-positive for PD-1, CD39, and / or TIGIT), the selected cell population is further expanded in a second expansion to obtain a second expanded cell population. In such examples, the second expanded T cell population is harvested as a therapeutic TIL composition. In some embodiments, the second expansion is carried out over a period of time to achieve a therapeutic dose. In some embodiments, the second expansion is carried out to achieve a cell expansion factor of at or about 500-fold to at or about 1000-fold, e.g., at or about 750-fold. In some embodiments, the second expansion is carried out for at or about 7 days to 21 days, e.g., for about 14 days. In some embodiments, the second expansion is carried out to achieve a therapeutic dose of at or about 500 million or more than at or about 500 million total cells.

[0119] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the input sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells secreting chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells surface-positive for C-X-C chemokine receptor type 5 (CXCR5) from the first expanded cell population to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, the second expanded T cell population being for use as a therapeutic cell composition. In some embodiments, the method comprises harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0120] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells that are surface-positive for PD-1, CD39, and / or TIGIT from the first expanded cell population to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, the second expanded T cell population being for use as a therapeutic cell composition. In some embodiments, the method comprises harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0121] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) processing a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimulators of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells that are surface positive for PD-1 and CD39 from the first expanded cell population to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimulators under conditions to generate a second expanded T cell population, the second expanded T cell population being for use as a therapeutic cell composition. In some embodiments, the method comprises harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0122] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) processing a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimulators of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells that are surface-positive for PD-1 and TIGIT from the first expanded cell population to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimulators under conditions to generate a second expanded T cell population, the second expanded T cell population being for use as a therapeutic cell composition. In some embodiments, the method comprises harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0123] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells that are surface-positive for CD39 and TIGIT from the first expanded cell population to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, the second expanded T cell population being for use as a therapeutic cell composition. In some embodiments, the method comprises harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0124] In some embodiments, provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells that are surface positive for PD-1, CD39, and TIGIT from the first expanded cell population to generate a selected population; and (d) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, the second expanded T cell population being for use as a therapeutic cell composition. In some embodiments, the method comprises harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0125] In some aspects of the provided methods, the methods do not include co-culturing a tumor-reactive T cell population with antigen-presenting cells that present one or more neo-antigenic peptides corresponding to associated non-synonymous somatic mutations in the subject's tumor.

[0126] In other aspects of the provided methods, the methods include co-culturing a tumor-reactive T cell population with antigen-presenting cells that present one or more neo-antigenic peptides corresponding to associated non-synonymous somatic mutations in the subject's tumor. In other such aspects of the provided methods, the methods include co-culturing T cells directly digested from a tumor fragment from the subject, or T cells derived from a tumor fragment from the subject after initial (e.g., minimal expansion), in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC), where the one or more non-native peptides correspond to associated non-synonymous somatic mutations in the subject's tumor. In some embodiments, prior to co-culturing, tumor-reactive T cells can be enriched or selected (e.g., based on the selection of cells positive for PD-1, CD39, and / or TIGIT), and the selected T cell population is co-cultured with antigen-presenting cells that present the peptide epitopes. Cells from the culture are then expanded to generate a therapeutic composition containing the expanded tumor-specific reactive cell population. In other embodiments, after co-culture, tumor-reactive T cells can be enriched or selected directly from the co-culture (e.g., based on the selection of cells positive for PD-1, CD39, and / or TIGIT), and the selected cells expanded to generate a therapeutic composition containing an expanded tumor-specific reactive cell population.

[0127] In some embodiments, the method includes (a) processing a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample comprising T cells; (b) performing a first expansion by culturing the input sample comprising T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting from the first expanded cell population cells that secrete chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells that are surface positive for C-X-C chemokine receptor type 5 (CXCR5) to generate a selected population; and (d) selecting from the first expanded cell population cells that secrete endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor. Provided herein are methods for producing tumor-reactive T cells, comprising: (a) co-culturing a selected population in the presence of antigen-presenting cells presenting one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells, wherein the one or more non-native peptides correspond to associated non-synonymous somatic mutations in the tumor of interest; and (b) performing a second expansion by culturing the reactive T cell population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. In some embodiments, the method includes harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0128] In some embodiments, the method includes (a) processing a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample comprising T cells; (b) performing a first expansion by culturing the input sample comprising T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; and (c) co-culturing the first expanded cell population in the presence of antigen-presenting cells that present one or more non-natural peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells comprising endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-natural peptides are stimuli of lymphocytes.

[0013] Provided herein are methods for producing tumor-reactive T cells, comprising the steps of: (a) selecting, from the reactive T cell population, cells secreting chemokine (C-X-C motif) ligand 13 (CXCL13) and / or cells surface-positive for C-X-C chemokine receptor type 5 (CXCR5) to generate a selected population; and (b) performing a second expansion by culturing the selected population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, the second expanded T cell population being for use as a therapeutic cell composition. In some embodiments, the method includes harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0129] (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) selecting cells from the first expanded cell population that are surface positive for PD-1, CD39, and / or TIGIT, e.g., PD-1, CD39, and TIGIT, to generate a selected cell population; (d) generating a reactive T cell population comprising T cells that contain endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor. Provided herein are methods for producing tumor-reactive T cells, the methods comprising: (a) co-culturing a selected cell population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to produce tumor-reactive T cells, wherein the one or more non-native peptides are peptides corresponding to associated non-synonymous somatic mutations in the tumor of interest; and (b) performing a second expansion by culturing the reactive T cell population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. In some embodiments, the method comprises harvesting the second expanded T cell population produced by the method for formulation as a therapeutic composition.

[0130] (a) treating a biological sample containing T cells obtained from a donor subject with a tumor to generate an input sample containing T cells; (b) performing a first expansion by culturing the sample containing T cells with one or more first T cell stimuli of lymphocytes under conditions to generate a first expanded T cell population; (c) co-culturing the first expanded cell population in the presence of antigen-presenting cells that present one or more non-natural peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells that contain endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-natural peptides are expressed. is a peptide corresponding to an associated non-synonymous somatic mutation in the subject's tumor; (d) selecting cells surface-positive for PD-1, CD39, and / or TIGIT, e.g., PD-1, CD39, and TIGIT, from the reactive T cell population to generate a selected cell population; and (e) performing a second expansion by culturing the selected cell population with one or more second T cell stimuli under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. In some embodiments, the method includes harvesting the second expanded T cell population generated by the method for formulation as a therapeutic composition.

[0131] In some embodiments, the T cell stimulator comprises anti-CD3 (e.g., an anti-CD3 antibody such as OKT3), an anti-CD28 reagent (e.g., an anti-CD28 antibody), e.g., an anti-CD3 antibody (e.g., OKT3) and an anti-CD28 antibody, and / or one or more recombinant cytokines (e.g., IL-2, IL-7, IL-21, and / or IL-15). In certain embodiments, the incubation or culture of the T cells is also performed using a nutrient-containing medium to enable the cells to survive outside the body.

[0132] In some cases, the method includes incubating with recombinant IL-2, alone or in combination with one or more other recombinant cytokines (e.g., IL-7, IL-21, and / or IL-15), and optionally one or more other T cell stimulators, to provide the cells with primary and secondary (co-stimulatory) signals. A standard method for culturing T cells and providing the cells with primary and secondary (co-stimulatory) signals includes incubation with T cell stimulators provided by anti-CD3 (e.g., OKT3) and anti-CD28 reagents. In some embodiments, the T cell stimulators include anti-CD3 antibodies (e.g., OKT3) and anti-CD28 antibodies. Typically, such stimulation also includes one or more additional recombinant cytokines (e.g., IL-2, IL-7, IL-21, and / or IL-15) and nutrient-containing medium to enable the cells to survive outside the body.

[0133] The provided methods for expanding tumor-reactive T cells include a first expansion that involves culturing a selected or isolated population containing T cells (i.e., a first T cell population) with recombinant cytokines, typically including recombinant IL-2 (e.g., IL-2, IL-7, IL-21, and / or IL-15). In some cases, the T cell stimulator further provides primary and secondary (co-stimulatory) signals to the cells, for example, provided by an anti-CD3 (e.g., OKT3) reagent and an anti-CD28 reagent, e.g., an anti-CD3 antibody (e.g., OKT3) and an anti-CD28 antibody. The initial or first expansion results in a second T cell population enriched for T cells as a result of the expansion or proliferation of T cells present in the first population.

[0134] In the provided methods, tumor-reactive T cells are further identified or enriched from the stimulated T cells expanded in the first step by one or more additional steps including ex vivo coculture of the stimulated T cells (second T cell population) with antigen-presenting cells (APCs) and one or more peptides comprising neoepitopes of tumor antigens (APC / peptide neoepitopes). In some embodiments, the provided methods include ex vivo coculture incubating the second T cell population with APCs, e.g., autologous APCs or artificial antigen-presenting cells (aAPCs), that have been exposed to or contacted with one or more peptides, e.g., synthetic peptides, under conditions in which the APCs are induced to present one or more peptides from tumor-associated antigens. In some embodiments, the T cell population is autologous T cells from a tumor-bearing subject, and the source of the synthetic peptide is a tumor antigen peptide derived from a tumor antigen of the same subject. In some embodiments, the cells obtained from the ex vivo coculture are a cell population (third population) comprising tumor-reactive T cells that recognize or are activated by peptides presented on the MHC of the APCs in the culture. In some embodiments, the cells obtained from the ex vivo co-culture are a source of cells enriched for tumor-reactive T cells.

[0135] In certain embodiments, the enriched or isolated T cells from the coculture are subjected to a second expansion, such as after separation or selection of tumor-reactive T cells or T cells that are surface-positive for one or more T cell activation markers associated with tumor-reactive T cells. The second expansion includes incubation with a T cell stimulator, such as an anti-CD3 antibody (e.g., OKT3), an anti-CD28 antibody, and a recombinant cytokine (e.g., IL-2, IL-7, IL-21, and / or IL-15), to further stimulate the T cells. T cells, e.g., 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, can be expanded for a certain number of days as desired and / or until the therapeutic dose or harvest dose is met. A composition of the expanded T cells can then be harvested and formulated for administration to a subject for the treatment of the subject's cancer.

[0136] In embodiments of the provided methods, one or more of the steps can be performed in serum-free medium. In one embodiment, the serum-free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcell technologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline (Sigma), Xvivo15 (Lonza), PrimeXV (Irvine Scientific), or Stem XVivo (RandD systems). The serum-free medium can be supplemented with a serum substitute, such as ICSR (immune cell serum replacement) from LifeTech. The level of serum substitute (e.g., ICSR) can be, for example, up to 5%, e.g., about 1%, about 2%, about 3%, about 4%, or about 5%. In some embodiments, the serum-free medium contains 0.5 mM to 5 mM of a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine (Glutamax™). In some embodiments, the concentration of the dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine, is 0.5 mM to 5 mM, 0.5 mM to 4 mM, 0.5 mM to 3 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, 1 mM to 4 mM, 1 mM to 3 mM, 1 mM to 2 mM, 2 mM to 5 mM, 2 mM to 4 mM, 2 mM to 3 mM, 3 mM to 5 mM, 3 mM to 4 mM, or In some embodiments, the concentration of a dipeptide form of L-glutamine, e.g., L-alanyl-L-glutamine, is 2 mM or about 2 mM.

[0137] In connection with the provided method, the method results in enrichment of T cells that contain endogenous TCR specific to tumor-associated antigens, so as to maximize the expansion of desired therapeutic cells.T cells, for example, tumor-reactive T cells, or T cells that are surface-positive for one or more T cell upregulation markers or activation markers associated with tumor-reactive T cells, can be expanded for a certain number of days as desired and / or until the 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 the treatment of the subject's cancer.

[0138] A. Stimulation of a T cell population to expand T cells, e.g., first expansion The provided methods include obtaining and enriching a T cell population from a biological sample for use as a first sample or input sample containing T cells. In some cases, the first sample or input sample of T cells is known to contain T cells reactive to tumor antigens or is likely to contain T cells reactive to tumor antigens, or can be reactive to tumor antigens after ex vivo co-culture with, for example, an autologous source of tumor antigen. For example, typically, the first sample or input sample of T cells is derived from a tumor or from a biological sample from a subject known to have or likely to have a tumor. In certain embodiments, the first sample or input sample of T cells is further stimulated with one or more T cell stimulators (e.g., one or more recombinant cytokines, such as IL-2) to generate a second T cell population or stimulated T cell population containing expanded T cells after stimulation.

[0139] In some embodiments, incubation with a T cell stimulator is performed directly on an input sample or first sample of T cells selected or obtained from a biological sample from the subject (e.g., autologous T cells from the subject), and the input sample or first sample of T cells is incubated with the T cell stimulator. In other embodiments, the input sample or first sample of T cells contains T cells that are likely or suspected to be tumor-reactive T cells, and such cells are first selected from a population of T cells selected from a biological sample from the subject by selecting cells positive for a marker associated with reactive T cells, e.g., one or more markers described in Section IC. In such embodiments, incubation with a T cell stimulator is performed after enriching the T cell population for tumor-reactive T cells. In provided embodiments, incubation with a T cell stimulator is performed prior to co-culture of such T cells with APC / peptide neoepitopes.

[0140] In some cases, the conditions for stimulating T cells by culture with one or more T cell stimulators result in activation of the cells and expansion or growth of T cells present in the first sample of T cells or input sample. In some embodiments, the conditions for stimulating T cells with one or more T cell stimulators can include culturing T cells under conditions that result in bulk expansion of T cells.

[0141] In the provided methods, the stimulated or expanded composition of T cells, e.g., the first expanded population, is then used in subsequent downstream processes for enrichment and expansion of tumor-reactive T cells, e.g., processes comprising co-culturing the stimulated T cells with antigen-presenting cells (APCs) in the presence of a T cell neoepitope (mutated) peptide antigen to generate, obtain, or remove T cells that are tumor-reactive T cells. In certain embodiments, the provided methods may also comprise a process of selecting or enriching T cells reactive to the tumor antigen (tumor-reactive T cells) after co-culturing the T cells with the APC / peptide neoepitope, e.g., by selecting cells positive for markers associated with reactive T cells, e.g., one or more markers described in Section IC. The tumor-reactive T cell population may be cultured under expansion conditions, e.g., to generate a therapeutic T cell composition.

[0142] In any aspect of the provided methods, the input sample or first sample of T cells is incubated in the presence of a T cell stimulator. In certain embodiments, the incubation is under conditions where the T cell stimulator activates or stimulates the cells or promotes expansion of T cells present in the input population of T cells, or the first population of T cells.

[0143] In some embodiments, the T cell stimulator comprises a recombinant T cell stimulatory cytokine such as IL-2, IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the T cell stimulatory cytokine comprises IL-2 alone or in combination with another cytokine from among IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the T cell stimulatory cytokine is one, two, three, or more of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the T cell stimulatory cytokine is IL-7 and IL-15.

[0144] In some embodiments, the T cell stimulator can include one or more agents involved in CD3. The T cell stimulator can include an anti-CD3 antibody, such as OKT3. In some embodiments, the T cell stimulator can further include an agent involved in CD3, such as an anti-CD28 agent (presented by APCs or as a soluble antibody). For example, the T cell stimulator can include an anti-CD3 antibody (e.g., OKT3) and an anti-CD28 antibody. Thus, in aspects of the provided methods, a first sample or input sample of T cells is incubated with one or more T cell stimulators of lymphocytes, such as, but not limited to, an anti-CD3 antibody (e.g., OKT3) and anti-CD28 (presented by APCs or as a soluble antibody), to generate a second T cell population containing activated or stimulated T cells. In some aspects, such stimulation can occur before co-culture in the presence of APCs and neoepitope (mutant) peptides. In certain embodiments, one or more recombinant cytokines are also present during the incubation as additional T cell stimulants.

[0145] In some embodiments, incubation with one or more T cell stimulators, e.g., anti-CD3 and / or recombinant cytokines, e.g., IL-2, can be continued for a period sufficient to activate or stimulate the cells. In some embodiments, incubation with the T cell stimulator is for 1 day or about 1 day, e.g., generally 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days, or for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days, or any range of time between any of the foregoing. In some embodiments, incubation is for 7-10 days. In some embodiments, incubation is for 7 days or about 7 days. In some embodiments, incubation is for 8 days or about 8 days. In some embodiments, incubation is for 9 days or about 9 days. In some embodiments, incubation is for 10 days or about 10 days. In some embodiments, incubation with the T cell stimulator is for between 12 and 96 hours, such as between 24 and 48 hours, typically at or about 48 hours.

[0146] In some embodiments, the cells are washed one or more times during culture to remove agents present during incubation or culture and / or to supplement the culture medium with one or more additional agents, hi some embodiments, the cells are washed during incubation or culture to reduce or remove T cell stimulatory agents prior to completion of culture.

[0147] In some embodiments, the methods of stimulating T cells provided herein include incubating with a T cell stimulator at a temperature suitable for proliferation of human T lymphocytes, e.g., at least about 25° C., generally at least about 30° C., generally at or about 37° C. In some embodiments, the culture or incubation method is carried out in serum-free medium.

[0148] 1. Samples containing T cells The provided methods include selecting or obtaining an input sample of T cells from a biological sample that can be used as a source or input of T cells for stimulation with one or more T cell stimulators (e.g., recombinant IL-2 or other T cell stimulatory cytokines and / or anti-CD3). In some embodiments, the T cells are derived from a biological sample from a subject known to contain or likely to contain tumor-reactive T cells. The collected biological sample contains or is suspected to contain lymphocytes with endogenous TCRs that are reactive to mutations present on the tumor.

[0149] In some aspects of the provided embodiments, a suitable biological sample is obtained from a subject, for example, a patient of interest, i.e., a patient suspected or known to have cancer. In some embodiments, the sample is a sample known or suspected to contain T cells, for example, T cells that may be specific to, bind to, or recognize tumor-associated antigens, or T cells that are likely to express the endogenous T cell receptor (TCR). The biological sample can be derived from any initial source that may contain such T cells or is suspected to contain such T cells. In some aspects, the source of the biological sample of interest includes, but is not limited to, many different physiological sources, such as tissue-derived samples, for example, homogenates, and blood or their derivatives.

[0150] Any of a variety of biological samples can be used as a source of potentially reactive T cells. Tumors and downstream lymph nodes may have the highest frequency of reactive T cells (Powell et al., Clin. Cancer. Res., 2014), but other sample sources can also be used. In some cases, the sample is a tumor sample, a tertiary lymphatic site, a draining lymph node, peripheral blood, or bone marrow. In some embodiments, the biological sample is a tumor sample. In some embodiments, the biological sample is a lymph sample. In some embodiments, the biological sample is a peripheral blood sample.

[0151] Biological samples include tissues, body fluids, and other samples taken directly from a subject to obtain an input sample, or may be subjected to one or more processing steps, such as separation, e.g., selection or enrichment, centrifugation, washing, and / or incubation. The input sample containing T cells may be a sample obtained directly from a biological source or a sample that has been processed. Biological samples include, but are not limited to, body fluids, e.g., blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, tissue and organ samples, and processed samples derived therefrom.

[0152] In some aspects, the sample is a blood or blood-derived sample, or an apheresis product or leukapheresis product, or is derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organ, and / or cells derived therefrom. In the context of cell therapy, e.g., adoptive cell therapy, samples include samples from autologous and allogeneic sources.

[0153] In many embodiments, the sample can be derived from a body fluid that is at least suspected to contain T cells of interest. In many embodiments, a suitable initial source for the sample is blood. In some embodiments, the biological sample is a blood-derived sample. The blood-derived sample can be derived from whole blood or a fraction thereof, such as serum, plasma, etc., and in many embodiments, the sample is derived from blood cells collected from whole blood. In some aspects, the sample source contains mononuclear cells. For example, the biological sample is or contains peripheral blood mononuclear cells (PBMCs), or is derived from PBMCs.

[0154] In some embodiments, the sample is a PBMC-derived sample, and the sample is generally a fluid PBMC-derived sample. Any convenient method for generating a fluid PBMC sample can be used. In many embodiments, the fluid PBMC-derived sample is prepared by separating PBMC from whole blood, that is, by collecting PBMC, for example, by centrifugation (for example, by Ficoll-Hypaque density gradient centrifugation, where representative protocols for such separation procedures are disclosed in WO98 / 15646 and U.S. Patent No. 5,985,565).

[0155] In some embodiments, the sample is a tumor sample, thereby providing a source of tumor-infiltrating lymphocytes (TILs). In some aspects, TILs are T cells that have left the subject's bloodstream and migrated into or infiltrated the tumor. In certain aspects, TILs are reactive to tumor antigens.

[0156] Patient tumor samples can be obtained by any of a variety of methods to obtain a sample containing a mixture of tumor cells and TIL cells. In some embodiments, tumor samples are obtained by surgical resection. In some embodiments, tumor samples are obtained by needle biopsy. Generally, tumor samples can be derived from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be derived from liquid tumors, e.g., tumors derived from hematological malignancies. Solid tumors can be any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach (gastrointestinal), and skin (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In certain embodiments, the tumor is any of those described in Section IV. In some embodiments, the tumor sample is derived from the same tumor source used to identify neoantigens for preparing peptide neoepitopes.

[0157] In provided embodiments, the tumor sample obtained is less than 1 mm in size. 3 or about 1 mm 3 From, 8mm 3 or about 8 mm 3 , e.g., 1 mm3 or about 1 mm 3 From, 6mm 3 or about 6 mm 3 , 1mm 3 or about 1 mm 3 From, 4mm 3 or about 4 mm 3 , 1mm 3 or about 1 mm 3 From, 2mm 3 or about 2 mm 3 In some embodiments, the tumor fragments are about 2-3 mm 3 In some embodiments, the tumor fragment is about 1-2 mm 3 In some embodiments, the tumor fragments are obtained by physical fragmentation, for example, by scraping. In some embodiments, the tumor fragments are obtained by sharp scraping.

[0158] In some of any of the provided embodiments, the obtained tumor sample is fragmented into pieces ranging from 1 mm or about 1 mm in diameter to 8 mm or about 8 mm in diameter, e.g., 1 mm or about 1 mm in diameter to 6 mm or about 6 mm in diameter, 1 mm or about 1 mm in diameter to 4 mm or about 4 mm in diameter, 1 mm or about 1 mm in diameter to 2 mm or about 2 mm in diameter. In some embodiments, the tumor fragments are about 2-3 mm in diameter. In some embodiments, the tumor fragments are about 1-2 mm in diameter. In some embodiments, the tumor fragments are obtained by physical fragmentation, e.g., by scraping. In some embodiments, the tumor fragments are obtained by sharp scraping.

[0159] In some embodiments, the tumor sample is cryopreserved prior to fragmentation. In some embodiments, the tumor fragments are cryopreserved.

[0160] In some embodiments, the resulting tumor fragments are used directly as input cells for T cells in the provided methods. In some embodiments, the resulting tumor fragments are placed in culture medium for stimulation of T cells under conditions for mediating T cell activation and maintaining T cell expansion, such as any of the conditions described in subsection IA2 below, and with appropriate nutrients for mediating T cell activation and maintaining T cell expansion. In some embodiments, 1 to 500 tumor fragments (e.g., 1 to 8 mm in size each) 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, plate, or other closed system device. In some embodiments, the culture vessel is a sealed vessel that provides a gas-permeable surface area, such as a gas-permeable flask. Exemplary culture vessels that provide a gas-permeable surface area include G-Rex plates or flasks. In some embodiments, one tumor fragment (approximately 1 to 8 mm in diameter) is placed in approximately 2 cm of the culture vessel. 2 The specific culture vessel can be selected based on the number of tumor fragments available and / or the desired yield of cells. The selection of the culture vessel (e.g., G-Rex) can be selected by linearly scaling the number of fragments seeded to the surface area of ​​the culture vessel. In some embodiments, the surface area of ​​the culture vessel is approximately 2 cm. 2 (e.g., a G-Rex 24-well plate), and about one tumor fragment (about 1-8 mm in diameter) is placed in the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 10 cm 2 (e.g., G-Rex 10 or G-Rex 10M), and about 5 tumor fragments (each about 1-8 mm in diameter) are placed in the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 100 cm. 2 (e.g., G-Rex 100 M / 100M-CS), and about 50 tumor fragments (each about 1-8 mm in diameter) are placed in the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 500 cm. 2(e.g., G-Rex 500 M / 500M-CS), in which approximately 250 tumor fragments (each approximately 1-8 mm in diameter) are placed in a culture vessel. In aspects of the provided methods, increasing the size of the culture vessel, and therefore the number of tumor fragments per vessel, compared to methods involving smaller culture vessels and / or fewer fragments per vessel can reduce variability, e.g., by pooling more fragments to minimize inter-tumor variability between fragments.

[0161] In some embodiments, the tumor fragments are placed in culture medium to stimulate the cells using any of the conditions described in subsection IA2 below. In some embodiments, the culture medium is serum-free medium containing recombinant cytokines such as IL-2, IL-7, IL-15, and / or IL-21, e.g., recombinant IL-12 or recombinant IL-7 and IL-15-derived cytokines. The concentrations of the recombinant cytokines may include any of those described. In particular embodiments, the culture medium is serum-free medium containing recombinant IL-2, e.g., from 300 IU / mL or about 300 IU / mL to 1000 IU / mL or about 1000 IU / mL, e.g., 300 IU / mL or about 300 IU / mL. In some embodiments, the culture medium is serum-free medium containing an anti-CD3 antibody and / or a CD28 targeting agent (e.g., an anti-CD28 antibody) and one or more recombinant cytokines (e.g., IL-2).

[0162] In some embodiments, tumor fragments are used as a source for preparing a single-cell suspension for use as an input sample for T cells in the provided methods. In some embodiments, the provided methods include obtaining cells from tumor fragments, e.g., by enzymatic digestion of the tumor fragments to obtain TILs. The enzymatic digestion can be performed using a collagenase, such as type IV collagenase or type I / II collagenase. The enzyme, such as collagenase, can be present in the medium for enzymatic digestion at a concentration of 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL, e.g., 1 mg / mL or about 1 mg / mL, 2 mg / mL or about 2 mg / mL, 3 mg / mL or about 3 mg / mL, 4 mg / mL or about 4 mg / mL, or 5 mg / mL or about 5 mg / mL, or any value between the foregoing. In some embodiments, the enzymatic digestion is performed with a medium containing, e.g., 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL of type IV collagenase. In some embodiments, enzymatic digestion is performed in a medium containing, for example, 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL of type I / II collagenase. In some embodiments, if a more gentle digestion is desired, 1 mg / mL or about 1 mg / mL of collagenase is used. In some embodiments, if a more thorough digestion is desired, a higher concentration of collagenase, for example, 5 mg / mL or about 5 mg / mL of collagenase, is used. In other embodiments, enzymes from the Miltenyi Human Tumor Dissociation Kit can be used (e.g., Cat. O. 130-095-929; Miltenyi Biotec). The enzyme-containing enzymatic medium can be a serum-free medium, such as any of those described. In certain embodiments, the enzyme medium comprises collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g., GlutaMAX), 10 mg / mL gentamicin, 30 units / mL DNase, and 1.0 mg / mL collagenase.In some embodiments, the enzyme medium includes serum-free medium (e.g., OpTmizer) containing 2 mM glutamate (e.g., GlutaMAX), 10 μg / mL gentamicin, immune cell serum replacement (e.g., CTS immune cell serum replacement), and 1.0 mg / mL to 5.0 mg / mL collagenase. In some embodiments, the collagenase is type IV collagenase. In some embodiments, the collagenase is type I / II collagenase.

[0163] The tumor fragments are then mechanically cut to dissociate the TILs, for example, using a tissue dissociator. An example of a tissue dissociator is the GentleMACs™ (Miltenyi Biotec) for homogenizing tissue. Tumor digests can be produced by placing the tumor in an enzyme medium, mechanically dissociating the tumor for about 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, followed by repeated cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments remain. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation can be performed using FICOLL to remove these cells. In some cases, separation can be achieved by centrifugation, in which case the cell pellet can be resuspended and strained, for example, through a 70 μm strainer to remove debris. Alternative methods known in the art can also be used, such as those described in U.S. Patent Application Publication No. 2012 / 0244133 A1, the disclosure of which is incorporated herein by reference. Any of the foregoing methods can be used in any of the embodiments described herein for methods of obtaining TILs for use in the provided methods.

[0164] In some embodiments, a single cell suspension for use as an input sample of T cells comprises 1 x 10 cells per gram of tumor sample from a subject. 6 pieces or approximately 1 x 10 6 T cells to 1000 x 10 per gram of subject-derived tumor sample 6 pieces or approximately 1000 x 10 6Up to 1 x 10 T cells per gram of tumor sample from a subject, e.g., 1 x 10 6 ~500×10 6 1 x 10 T cells per gram of subject-derived tumor sample 6 ~100×10 6 1 x 10 T cells per gram of subject-derived tumor sample 6 ~50×10 6 1 x 10 T cells per gram of subject-derived tumor sample 6 ~10×10 6 10 x 10 T cells per gram of subject-derived tumor sample 6 ~1000×10 6 10 x 10 T cells per gram of subject-derived tumor sample 6 ~100×10 6 10 x 10 T cells per gram of subject-derived tumor sample 6 ~500×10 6 10 x 10 T cells per gram of subject-derived tumor sample 6 ~50×10 6 50 x 10 T cells per gram of subject-derived tumor sample 6 ~1000×10 6 50 x 10 T cells per gram of subject-derived tumor sample 6 ~500×10 6 50 x 10 T cells per gram of subject-derived tumor sample 6 ~100×10 6 100 x 10 T cells per gram of subject-derived tumor sample 6 ~1000×10 6 100 x 10 T cells per gram of subject-derived tumor sample 6 ~500×10 6 T cells or 500 x 10 per gram of tumor sample from a subject 6 ~1000×10 6 In some embodiments, a single cell suspension for use as an input sample of T cells comprises 10 x 10 T cells per gram of tumor sample from a subject. 6 T cells, 20 x 10 6 T cells, 30 x 10 6 T cells, 40 x 106 T cells, 50 x 10 6 T cells, 60 x 10 6 T cells, 70 x 10 6 T cells, 80 x 10 6 T cells, 90 x 10 6 T cells, 100 x 10 6 T cells, or approximately 10 x 10 6 T cells, 20 x 10 6 T cells, 30 x 10 6 T cells, 40 x 10 6 T cells, 50 x 10 6 T cells, 60 x 10 6 T cells, 70 x 10 6 T cells, 80 x 10 6 T cells, 90 x 10 6 T cells, 100 x 10 6 T cells, or at least 10 x 10 6 T cells, 20 x 10 6 T cells, 30 x 10 6 T cells, 40 x 10 6 T cells, 50 x 10 6 T cells, 60 x 10 6 T cells, 70 x 10 6 T cells, 80 x 10 6 T cells, 90 x 10 6 T cells, 100 x 10 6 T cells, or at least about 10 x 10 6 T cells, 20 x 10 6 T cells, 30 x 10 6 T cells, 40 x 10 6 T cells, 50 x 10 6 T cells, 60 x 10 6 T cells, 70 x 10 6 T cells, 80 x 10 6 T cells, 90 x 10 6 T cells, 100 x 10 6 In some embodiments, a single cell suspension for use as an input sample of T cells comprises 10 x 10 T cells per gram of tumor sample from a subject. 6 pieces or approximately 10 x 106 to 100 x 10 per gram of subject-derived tumor sample 6 pieces or approximately 100 x 10 6 Contains T cells.

[0165] In some embodiments, digested cells from tumor fragments are placed in culture medium as a single cell suspension under conditions for mediating T cell activation and / or T cell expansion, such as any of the conditions described in subsection IA2 below, for stimulation of T cells, along with appropriate nutrients for mediating T cell activation and / or T cell expansion. Cells are seeded at a specific density appropriate for the particular culture vessel. The culture vessel can be a microwell, flask, tube, bag, or other closed system device. In some embodiments, the culture vessel is a sealed vessel providing a gas permeable surface area, such as a gas permeable flask. Exemplary culture vessels providing a gas permeable surface area include G-Rex plates or flasks. In some embodiments, cells are seeded at a density of approximately 5 x 10 5 ~2×10 6 The enzymatically digested single cell suspension of 100 cells was placed in approximately 2 cm of the culture vessel. 2 The specific culture vessel can be selected based on the number of cells available and / or the desired yield of cells. The selection of the culture vessel (e.g., G-Rex) can be selected by linearly scaling the number of cells seeded to the surface area of ​​the culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 2 cm. 2 (e.g., G-Rex 24-well plate), and approximately 5 × 10 5 ~2×10 6 The enzymatically digested single cell suspension of cells is placed in a culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 10 cm. 2 (e.g., G-Rex 10 or G-Rex 10M), which is approximately 2.5 × 10 6 ~1×10 7 The enzymatically digested single cell suspension of cells is placed in a culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 100 cm. 2 (e.g., G-Rex 100 M / 100M-CS), which is approximately 2.5 × 10 7~1×10 8 The enzymatically digested single cell suspension of cells is placed in a culture vessel. In some embodiments, the surface area of ​​the culture vessel is about 500 cm. 2 (e.g., G-Rex 500 M / 500M-CS), which is approximately 1.25 × 10 8 ~5×10 8 An enzymatically digested single cell suspension of 100 cells is placed in a culture vessel.

[0166] In some embodiments, the culture medium is a serum-free medium containing recombinant IL-2. In some embodiments, one or more additional T cell stimulatory substances can also be included. In some embodiments, the culture medium is a serum-free medium containing an anti-CD3 antibody and / or a CD28 targeting agent (e.g., an anti-CD28 antibody) and one or more recombinant cytokines (e.g., IL-2, IL-7, IL-15, and / or IL-21).

[0167] Samples can be obtained from a variety of different subjects / patients / hosts. Generally, such hosts are "mammals" or "mammals," which terms are used broadly to refer to organisms belonging to the class Mammalia, including Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the host is human.

[0168] In some aspects, the subject is human.Thus, in some embodiments, the cell is a primary cell, for example, a primary human cell.In some embodiments, the sample is autologous to the subject to be treated, for example, the subject is a patient who needs a specific therapeutic intervention, such as adoptive cell therapy, in which cells are isolated, processed and / or expanded according to the provided method.In some embodiments, the sample is allogeneic to the subject to be treated.

[0169] In some of the provided embodiments, the biological sample is a lymphoid-derived sample or a tumor-derived sample, and the number of cells at the start of the culturing step is 10×10 6 pieces or approximately 10 x 10 6From 100 x 10 6 pieces or approximately 100 x 10 6 Total viable cells, 20 x 10 6 pieces or approximately 20 x 10 6 From 100 x 10 6 pieces or approximately 100 x 10 6 total viable cells, or 12 x 10 6 pieces or approximately 12 x 10 6 From 43 x 10 6 Pieces or approximately 43 x 10 6 total viable cells or 10 x 10 6 pieces or approximately 10 x 10 6 Total viable cells, 12 x 10 6 pieces or approximately 12 x 10 6 Total viable cells, 20 x 10 6 Total viable cells, 40 x 10 6 Total viable cells, 60 x 10 6 total viable cells, or 100 x 10 6 In some embodiments, the percentage of tumor-reactive T cells at the start of the culturing step is from 1% or about 1% to 90% or about 90%, from 1% or about 1% to 75% or about 75%, from 1% or about 1% to 50% or about 50%, from 1% or about 1% to 25% or about 25%, or from 1% or about 1% to 14% or about 14%.

[0170] 2. Stimulation of T cells for initial expansion In aspects of the provided methods, T cells from an input sample (e.g., an input population of T cells, or a first T cell population, present in a resected tumor fragment, or a single-cell suspension from the resected tumor fragment) are incubated or cultured under conditions to stimulate the T cells, e.g., in the presence of one or more T cell stimulators, to expand the T cells. In some embodiments, incubation or culture with one or more T cell stimulators results in expansion (first expansion) or growth of selected T cells, or a desired subset or subtype thereof, or live cells thereof, for use in subsequent steps of the provided methods. Non-limiting examples of T cell stimulators and conditions for incubation or culture are described herein.

[0171] Thus, among the methods provided are methods of culturing T cells to produce tumor-reactive T cells, wherein the T cells are cultured or incubated in the presence of a T cell stimulator under conditions to expand the T cells.

[0172] In some embodiments, the T cell stimulator comprises a recombinant T cell stimulatory cytokine such as IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the T cell stimulatory cytokine comprises IL-2 alone or in combination with another cytokine from among IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the T cell stimulator comprises a recombinant T cell stimulatory cytokine such as IL-2, IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the T cell stimulatory cytokine comprises IL-2 alone or in combination with another cytokine from among IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the T cell stimulatory cytokines are one, two, three, or more of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the T cell stimulatory cytokines include 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 stimulatory cytokines include IL-2 alone or in combination with another cytokine from among IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the T cell stimulatory cytokines are IL-7 and IL-15.

[0173] In some embodiments, the selection of a cytokine or combination of cytokines is within the level of one skilled in the art, as long as the cytokine or cytokines provide the activity of stimulating and expanding T cells. The activity of stimulating tumor-reactive T cells can be direct or indirect. In some embodiments, one or more cytokines directly stimulate tumor-reactive T cells to expand or proliferate. In some embodiments, one or more cytokines suppress T regulatory T cells, thereby indirectly stimulating or enhancing the proliferation of desired tumor-reactive T cells.

[0174] In some embodiments, incubation with a T cell stimulator for initial expansion does not include incubation with one or more agents involving CD3 and costimulatory molecules, e.g., CD28. In some embodiments, incubation with a T cell stimulator for initial expansion does not include incubation with an anti-CD3 antibody, such as OKT3. In some embodiments, incubation with a T cell stimulator for initial expansion does not include incubation with an anti-CD3 (e.g., OKT3) / anti-CD28 antibody presented by APCs, either immobilized on a solid surface (e.g., beads) or as a soluble antibody. In some embodiments, incubation with a T cell stimulator for initial expansion does not include incubation with a soluble anti-CD3, such as OKT3. In some embodiments, incubation with a T cell stimulator for initial expansion does not include incubation with an anti-CD3 / anti-CD28 containing reagent immobilized on beads, such as those provided by Dynabeads. In some embodiments, incubation with a T cell stimulator for initial expansion does not include incubation with APCs, such as irradiated APCs, hi some embodiments, incubation with a T cell stimulator for initial expansion does not include incubation with non-dividing PBMCs, such as irradiated PBMCs.

[0175] In some of any of the provided embodiments, the T cell stimulatory agent is selected from an agent that initiates TCR / CD3 intracellular signaling and an agent that initiates signaling through a costimulatory receptor. In some of 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 through a costimulatory receptor comprises peripheral blood mononuclear cells (PBMCs), optionally non-dividing PBMCs or irradiated PBMCs. In some of any of the provided embodiments, the agent that initiates signaling through a costimulatory receptor is an anti-CD28 antibody. In some of any of the provided embodiments, the T cell stimulatory agents are soluble anti-CD3 and anti-CD28 antibodies, respectively. In certain embodiments, one or more recombinant cytokines are also present as additional T cell stimulatory agents during incubation. In some embodiments, incubation with a T cell stimulator comprises incubation with at least one T cell stimulatory recombinant cytokine (e.g., recombinant IL-2, IL-7, IL-21, IL-15, IL-25, IL-23, IL-27, and / or IL-35), and an additional T cell stimulator that engages CD3 and / or a costimulatory molecule (e.g., CD28) on the T cells.

[0176] In embodiments of the provided methods, the stimulatory conditions include one or more agents, e.g., ligands, that turn on or initiate the TCR / CD3 intracellular signaling cascade in the T cell and / or a costimulatory signal in the T cell. Such agents can include antibodies, e.g., those specific for TCR components, e.g., anti-CD3 and / or costimulatory receptors, e.g., anti-CD28 or anti-4-1BB. In some embodiments, such agents are added to the culture medium as soluble antibodies. In other embodiments, such agents are bound to a solid support, such as beads. In some embodiments, the T cell stimulatory agent comprises anti-CD3 / CD28-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0177] Anti-CD3 antibodies can include any antibody that is directed against or can specifically bind to the CD3 receptor on the surface of T cells, typically human CD3 on human T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include UHCTI clones, also known as T3 and CD3E. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. Anti-CD3 antibodies can be added as a soluble reagent or can be bound to beads. In certain embodiments, the anti-CD3 antibody is soluble.

[0178] In certain embodiments, the T cell stimulator comprises an anti-CD3 antibody that is added to the cell culture medium during incubation. In some embodiments, the anti-CD3 antibody has a concentration of from 0.1 ng / mL or about 0.1 ng / mL to 50 ng / mL, e.g., from 0.5 ng / mL or about 0.5 ng / mL to 50 ng / mL or about 50 ng / mL, from 0.5 ng / mL or about 0.5 ng / mL to 30 ng / mL or about 30 ng / mL, from 0.5 ng / mL or about 0.5 ng / mL to 15 ng / mL or about 15 ng / mL, from 0.5 ng / mL or about 0.5 ng / mL to 5 ng / mL or about 5 ng / mL, from 0.5 ng / mL or about 0.5 ng / mL to 1 ng / mL or about 1 ng / mL, from 1 ng / mL or about 1 ng / mL to 50 ng / mL or about 50 ng / mL, from 1 ng / mL or about 1 ng / mL to 30 ng / mL or about 30 ng / mL 50ng / mL or about 50ng / mL, 5ng / mL or about 5ng / mL to 30ng / mL or about 30ng / mL, 5ng / mL or about 5ng / mL to 15ng / mL or about 15ng / mL, 15ng / mL or about 15ng / mL to 50ng / mL or about 50ng / mL, 5ng / mL or about 5ng / mL to 30ng / mL or about 30ng / mL, 5ng / mL or about 5ng / mL to 15ng / mL or about 15ng / mL, 15ng / mL or about 15ng / mL to 50ng / mL or about 50ng / mL, 15ng / mL or about 15ng / mL to 30ng / mL or about 30ng / mL, or 30ng / mL or about 30ng / mL to 50ng / mL or about 50ng / mL, inclusive.

[0179] In a specific embodiment, the anti-CD3 antibody is OKT3. In one 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 the OKT3 antibody. In one embodiment, the cell culture medium comprises 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL of OKT3 antibody.

[0180] In some embodiments, the T cell stimulator comprises incubation with an anti-CD3 antibody and an additional agent that specifically binds to CD28 or stimulates or induces a CD28-mediated signal within the cell. In some embodiments, the CD28-mediated signal can be initiated or provided by an anti-CD28 antibody or an 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 (PBMCs).

[0181] In some embodiments, the T cell stimulator can include adding T cell feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the population. In some aspects, the non-dividing feeder cells can include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000-3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium prior to addition of the T cell population. In some embodiments, the resulting cell population 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 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, about 1:400, or about 1:500.

[0182] In some embodiments, stimulation does not include incubation with PBMCs or other feeder cells, eg, non-dividing or irradiated PBMCs or other non-dividing or irradiated feeder cells.

[0183] In some embodiments, the T cell stimulator may include adding an anti-CD28 antibody or an antigen-binding fragment thereof to the cell population. The anti-CD28 antibody may include any antibody directed against or capable of specifically binding to 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 may be added as a soluble reagent or bound to beads. In certain embodiments, the anti-CD3 antibody is soluble. In some embodiments, the anti-CD28 antibody has a concentration of from 1 ng / mL or about 1 ng / mL, 1000 ng / mL, 1 ng / mL or about 1 ng / mL, 500 ng / mL, 1 ng / mL or about 1 ng / mL, 100 ng / mL or about 100 ng / mL, 1 ng / mL or about 1 ng / mL, 10 ng / mL or about 10 ng / mL, 10 ng / mL or about 10 ng / mL, 1000 ng / mL or about 1000 ng / mL, 10 ng / mL or about 10 ng / mL to 500ng / mL or about 500ng / mL, 10ng / mL or about 10ng / mL to 100ng / mL or about 100ng / mL, 100ng / mL or about 100ng / mL to 1000ng / mL or about 1000ng / mL, 100ng / mL or about 100ng / mL to 500ng / mL or about 500ng / mL, or 500ng / mL or about 500ng / mL to 1000ng / mL or about 1000ng / mL.

[0184] In some embodiments, the T cell stimulator comprises one or more recombinant cytokines. In some embodiments, the cytokines are added to the culture medium or are exogenous to the culture medium. Thus, in some embodiments, one or more additional recombinant cytokines are also included in the culture. In some embodiments, the recombinant cytokines may comprise one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the recombinant cytokines may comprise one or more of IL-2, IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the culturing and incubation is performed in the presence of recombinant IL-2, IL-15, and IL-7. In some embodiments, the culturing is performed in the presence of IL-2. In some embodiments, the culturing is performed in the presence of IL-15 and IL-17, and in some aspects does not further comprise IL-2. In certain embodiments, the recombinant cytokines are human.

[0185] The recombinant cytokine is generally a recombinant human protein. In certain embodiments, the recombinant cytokine is present in the cell culture medium during incubation at a concentration of at least 0.5 IU / mL or at least about 0.5 IU / mL, at least 1.0 IU / mL or at least about 1.0 IU / mL, at least 5 IU / mL or at least about 5 IU / mL, at least 10 IU / mL or at least about 10 IU / mL, at least 100 IU / mL or at least about 100 IU / mL. or about 100 IU / mL, at least 1000 IU / mL or at least about 1000 IU / mL or 1000 IU / mL or about 1000 IU / mL, at least 1500 IU / mL or at least about 1500 IU / mL or 1500 IU / mL or about 1500 IU / mL, at least 2000 IU / mL or at least about 2000 IU / mL or 2000 IU / mL or about 2000 IU / mL, at least 2500 IU / mL or at least about 2500 IU / mL at least 2500 IU / mL or about 2500 IU / mL, at least 3000 IU / mL or at least about 3000 IU / mL or 3000 IU / mL or about 3000 IU / mL, at least 3500 IU / mL or at least about 3500 IU / mL or 3500 IU / mL or about 3500 IU / mL, at least 4000 IU / mL or at least about 4000 IU / mL or 4000 IU / mL or about 4000 IU / mL, at least 4500 IU / mL or or at least about 4500 IU / mL or 4500 IU / mL or about 4500 IU / mL, at least 5000 IU / mL or at least about 5000 IU / mL or 5000 IU / mL or about 5000 IU / mL, at least 5500 IU / mL or at least about 5500 IU / mL or 5500 IU / mL or about 5500 IU / mL, at least 6000 IU / mL or at least about 6000 IU / mL or 6000 IU / mL or about 6000 IU / mL,It is present in a concentration of at least or at least about 6500 IU / mL or 6500 IU / mL or about 6500 IU / mL, at least 7000 IU / mL or at least about 7000 IU / mL or 7000 IU / mL or about 7000 IU / mL, at least 7500 IU / mL or at least about 7500 IU / mL or 7500 IU / mL or about 7500 IU / mL, or at least 8000 IU / mL or at least about 8000 IU / mL or 8000 IU / mL or about 8000 IU / mL. In one embodiment, the cell culture medium comprises from 10 IU / mL or about 10 IU / mL, 100 IU / mL or about 100 IU / mL, 100 IU / mL or about 100 IU / mL, 1000 IU / mL or about 1000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 3000 IU / mL or about 3000 IU / mL, 3000 IU / mL or about 3000 IU / mL, 40 00 IU / mL or about 4000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 5000 IU / mL or about 5000 IU / mL, 5000 IU / mL or about 5000 IU / mL, 6000 IU / mL or about 6000 IU / mL, 6000 IU / mL or about 6000 IU / mL, 7000 IU / mL or about 7000 IU / mL, 7000 IU / mL or about 7000 IU / mL, 8000 IU / mL or about 8000 IU / mL (inclusive).

[0186] In some embodiments, recombinant IL-2 is present in the cell culture medium. In some aspects, IL-2 is the only recombinant cytokine added to the culture. In some aspects, recombinant IL-2 and one other recombinant regulatory cytokine from IL-7, IL-15, IL-21, IL-23, IL-25, IL-27, or IL-35 are added to the culture. IL-2 is a cytokine that supports T cell recovery and proliferation. IL-2 also supports T cell homeostasis, thereby supporting their phenotype, differentiation state, and immune memory. In some cases, inducing regulatory T cells within the tumor microenvironment can result in low bioavailability of IL-2. Recombinant IL-2 is routinely used for the widespread expansion of T cells in various settings. Recombinant IL-2 is commercially available. In certain embodiments, the recombinant IL-2 is GMP grade (e.g., MACS GMP Recombinant Human IL-2, Miltenyi Biotec).

[0187] Recombinant IL-2 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-2 can be included in the initial T cell expansion (first expansion), for example, to promote the growth of TILs and allow their proliferation from solid tumors. IL-2 can also be included in antigen-presenting cell coculture, as described in Section IB2, for example, to allow peak activation of neoantigen-reactive T cells prior to their isolation or selection. In some cases, recombinant IL-2 can also be included in the culture to expand tumor-reactive T cells during the second expansion phase, for example, as described in Section IB.

[0188] In some embodiments, the recombinant IL-2 is at or about 10 IU / mL to 1000 IU / mL, e.g., 10 IU / mL or about 10 IU / mL to 600 IU / mL or about 600 IU / mL, 10 IU / mL or about 10 IU / mL to 400 IU / mL or about 400 IU / mL, 10 IU / mL or about 10 IU / mL to 200 IU / mL or about 200 IU / mL, 10 IU / mL or about 10 IU / mL to 100 IU / mL or about 1 ... U / mL or about 10 IU / mL to 50 IU / mL or about 50 IU / mL, 50 IU / mL or about 50 IU / mL to 1000 IU / mL or about 1000 IU / mL, 50 IU / mL or about 50 IU / mL to 600 IU / mL or about 600 IU / mL, 50 IU / mL or about 50 IU / mL to 400 IU / mL or about 400 IU / mL, 50 IU / mL or about 50 IU / mL to 200 IU / mL or about 200 IU / mL, 50 IU / mL or about 50 IU / mL to 100 IU / mL or or about 100 IU / mL, 100 IU / mL or about 100 IU / mL, 1000 IU / mL or about 1000 IU / mL, 100 IU / mL or about 100 IU / mL, 600 IU / mL or about 600 IU / mL, 100 IU / mL or about 100 IU / mL, 400 IU / mL or about 400 IU / mL, 100 IU / mL or about 100 IU / mL, 200 IU / mL or about 200 IU / mL, 200 IU / mL or about 200 IU / mL, 1000 IU / mL or about 1000 IU / mL, 20 It is added to the culture medium at a concentration of from 0 IU / mL or about 200 IU / mL, 600 IU / mL or about 600 IU / mL, 200 IU / mL or about 200 IU / mL, 400 IU / mL or about 400 IU / mL, 400 IU / mL or about 400 IU / mL, 1000 IU / mL or about 1000 IU / mL, 400 IU / mL or about 400 IU / mL, 600 IU / mL or about 600 IU / mL, or 600 IU / mL or about 600 IU / mL, 1000 IU / mL or about 1000 IU / mL.In some embodiments, the recombinant IL-2 is present in an amount of 50-400 IU / mL.

[0189] In some embodiments, the initial expansion is carried out in the presence of recombinant IL-2 added at a concentration of 200 IU / mL to 1000 IU / mL or about 1000 IU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 200 IU / mL or about 200 IU / mL, 300 IU / mL or about 300 IU / mL, 400 IU / mL or about 400 IU / mL, 500 IU / mL or about 500 IU / mL, 600 IU / mL or about 600 IU / mL, 700 IU / mL or about 700 IU / mL, 800 IU / mL or about 900 IU / mL, 1000 IU / mL or about 1000 IU / mL, or any concentration between any of the foregoing. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 300 IU / mL or about 300 IU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 600 IU / mL or about 600 IU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 1000 IU / mL or about 1000 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine from IL-7, IL-15, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0190] In some embodiments, the incubation is performed with an even higher dose of IL-2. In some aspects, IL-2 is the only recombinant cytokine added to the culture medium.

[0191] In some embodiments, the recombinant IL-2 is administered at a concentration of from 1000 IU / mL or about 1000 IU / mL, to 8000 IU / mL or about 8000 IU / mL, e.g., from 1000 IU / mL or about 1000 IU / mL, to 7000 IU / mL or about 7000 IU / mL, from 1000 IU / mL or about 1000 IU / mL, to 6000 IU / mL or about 6000 IU / mL, from 1000 IU / mL or about 1000 IU / mL, to 5000 IU / mL or about 5000 IU / mL, from 1000 IU / mL or about 1000 IU / mL, From 4000 IU / mL or about 4000 IU / mL, 1000 IU / mL or about 1000 IU / mL, from 2000 IU / mL or about 2000 IU / mL, from 2000 IU / mL or about 2000 IU / mL, from 8000 IU / mL or about 8000 IU / mL, from 2000 IU / mL or about 2000 IU / mL, from 7000 IU / mL or about 7000 IU / mL, from 2000 IU / mL or about 2000 IU / mL, from 6000 IU / mL or about 6000 IU / mL, from 2000 IU / mL or about 2000 IU / mL, from 5000 IU / mL or about 5000 IU / mL, 2000 IU / mL or about 2000 IU / mL, from 4000 IU / mL or about 4000 IU / mL, from 4000 IU / mL or about 4000 IU / mL, from 8000 IU / mL or about 8000 IU / mL, from 4000 IU / mL or about 4000 IU / mL, from 7000 IU / mL or about 7000 IU / mL, from 4000 IU / mL or about 4000 IU / mL, from 6000 IU / mL or about 6000 IU / mL, from 4000 IU / mL or about 4000 IU / mL, from 5000 IU / mL or about 5000 IU / mL, from 5000 IU / mL or about 5000 IU / mL, from 8000 IU / mL or about 8000 IU / mL, from 5000 IU / mL or about 5000 IU / mL, from 7000 IU / mL or about 7000 IU / mL, from 5000 IU / mL or about 5000 IU / mL, from 6000 IU / mL or about 6000 IU / mL, from 6000 IU / mL or about 6000 IU / mL, from 8000 IU / mL or about 8000 IU / mL, from 6000 IU / mL or about 6000 IU / mL,The recombinant IL-2 is added to the culture medium at a concentration of 7000 IU / mL or about 7000 IU / mL, or from 7000 IU / mL or about 7000 IU / mL to 8000 IU / mL or about 8000 IU / mL. In some embodiments, the recombinant IL-2 is present in an amount of 6000 IU / mL or about 6000 IU / mL.

[0192] In some embodiments, recombinant IL-15 is present in the cell culture medium. IL-15 is a cytokine involved in the homeostasis and activation of memory T cells. In some cases, IL-15 can promote the effector function 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 certain embodiments, the recombinant IL-15 is GMP grade (e.g., MACS GMP Recombinant Human IL-15, Miltenyi Biotec).

[0193] Recombinant IL-15 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-15 can be included in the initial T cell expansion (first expansion), e.g., to promote the expansion of TILs to promote their growth, allow their proliferation, and / or stabilize the phenotype from solid tumors. Recombinant IL-15 can also be included in antigen-presenting cell cocultures, as described in Section IB2, e.g., to allow peak activation of neoantigen-reactive T cells prior to their isolation or selection. In some cases, recombinant IL-15 can also be included in the culture to expand tumor-reactive T cells during the second expansion phase, as described in Section IB. In some cases, recombinant IL-15 and recombinant IL-7 can be combined in the provided methods to provide activation, survival, and / or expansion of tumor-reactive T cells. In some such embodiments, the combination of recombinant IL-7 and recombinant IL-15 is a substitute for the use of recombinant IL-2 in the culture, and the culture medium does not further contain recombinant IL-2.

[0194] In some embodiments, recombinant IL-15 is administered at a concentration of from 10 IU / mL or about 10 IU / mL to 500 IU / mL, e.g., from 10 IU / mL or about 10 IU / mL to 400 IU / mL or about 400 IU / mL, from 10 IU / mL or about 10 IU / mL to 300 IU / mL or about 300 IU / mL, from 10 IU / mL or about 10 IU / mL to 200 IU / mL or about 200 IU / mL, from 10 IU / mL or about 10 IU / mL to 100 IU / mL or about 100 IU / mL, from 10 IU / mL or about 10 IU / mL from 70 IU / mL or about 70 IU / mL, from 10 IU / mL or about 10 IU / mL, from 50 IU / mL or about 50 IU / mL, from 10 IU / mL or about 10 IU / mL, from 30 IU / mL or about 30 IU / mL, from 30 IU / mL or about 30 IU / mL, from 500 IU / mL, from 30 IU / mL or about 30 IU / mL, from 400 IU / mL or about 400 IU / mL, from 30 IU / mL or about 30 IU / mL, from 300 IU / mL or about 300 IU / mL, from 30 IU / mL or about 30 IU / mL, From 00 IU / mL or about 200 IU / mL, 30 IU / mL or about 30 IU / mL, 100 IU / mL or about 100 IU / mL, 30 IU / mL or about 30 IU / mL, 70 IU / mL or about 70 IU / mL, 30 IU / mL or about 30 IU / mL, 50 IU / mL or about 50 IU / mL, 50 IU / mL or about 50 IU / mL, 400 IU / mL or about 400 IU / mL, 50 IU / mL or about 50 IU / mL, 500 IU / mL or about 500 IU / mL, 50 IU / mL or about from 50 IU / mL to 300 IU / mL or about 300 IU / mL, from 50 IU / mL or about 50 IU / mL to 200 IU / mL or about 200 IU / mL, from 50 IU / mL or about 50 IU / mL to 100 IU / mL or about 100 IU / mL, from 50 IU / mL or about 50 IU / mL to 70 IU / mL or about 70 IU / mL, from 70 IU / mL or about 70 IU / mL to 500 IU / mL or about 500 IU / mL, from 70 IU / mL or about 70 IU / mL to 400 IU / mL or about 400 IU / mL,From 70 IU / mL or about 70 IU / mL, 300 IU / mL or about 300 IU / mL, 70 IU / mL or about 70 IU / mL, 200 IU / mL or about 200 IU / mL, 70 IU / mL or about 70 IU / mL, 100 IU / mL or about 100 IU / mL, 100 IU / mL or about 100 IU / mL, 500 IU / mL or about 500 IU / mL, 100 IU / mL or about 100 IU / mL, 400 IU / mL or about 400 IU / mL, 100 IU / mL or about 100 IU / mL, 300 IU / mL or about 300 IU / mL, 100 IU / mL or about 100 IU / mL, 200 IU / mL or or about 200 IU / mL, 200 IU / mL or about 200 IU / mL, 500 IU / mL or about 500 IU / mL, 200 IU / mL or about 200 IU / mL, 400 IU / mL or about 400 IU / mL, 200 IU / mL or about 200 IU / mL, 300 IU / mL or about 300 IU / mL, 300 IU / mL or about 300 IU / mL, 500 IU / mL or about 500 IU / mL, 200 IU / mL or about 200 IU / mL, 400 IU / mL or about 400 IU / mL, or 400 IU / mL or about 400 IU / mL, 500 IU / mL or about 500 IU / mL. In some embodiments, IL-15 is added to the culture medium in an amount of 100 IU / mL or about 100 IU / mL to 200 IU / mL or about 200 IU / mL. In some embodiments, IL-15 is added to the culture medium at 180 IU / mL or about 180 IU / mL.

[0195] In some embodiments, the incubation is performed with an even higher dose of IL-15.

[0196] In some embodiments, the recombinant IL-15 is administered at a concentration of from 500 IU / mL or about 500 IU / mL to 5000 IU / mL or about 5000 IU / mL, e.g., from 500 IU / mL or about 500 IU / mL to 4000 IU / mL or about 4000 IU / mL, from 500 IU / mL or about 500 IU / mL to 2000 IU / mL or about 2000 IU / mL, from 500 IU / mL or about 500 IU / mL to 1500 IU / mL or about 1500 IU / mL, from 500 IU / mL or about 500 IU / mL to 1000 IU / mL. / mL or about 1000 IU / mL, 500 IU / mL or about 500 IU / mL, 750 IU / mL or about 750 IU / mL, 750 IU / mL or about 750 IU / mL, 5000 IU / mL or about 5000 IU / mL, 750 IU / mL or about 750 IU / mL, 4000 IU / mL or about 4000 IU / mL, 750 IU / mL or about 750 IU / mL, 2000 IU / mL or about 2000 IU / mL, 750 IU / mL or about 750 IU / mL, 1500 IU / mL or From about 1500 IU / mL, 750 IU / mL or about 750 IU / mL, 1000 IU / mL or about 1000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 5000 IU / mL or about 5000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 1500 IU / mL or from about 1500 IU / mL, 1500 IU / mL or about 1500 IU / mL, 5000 IU / mL or about 5000 IU / mL, 1500 IU / mL or about 1500 IU / mL, 4000 IU / mL or about 4000 IU / mL, 1500 IU / mL or about 1500 IU / mL, 2000 IU / mL or about 2000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 5000 IU / mL or about 5000 IU / mL, for example, from 2000 IU / mL or about 2000 IU / mL,In some embodiments, recombinant IL-15 is added to the culture medium at a concentration of 500 IU / mL or about 500 IU / mL, 600 IU / mL or about 600 IU / mL, 700 IU / mL or about 700 IU / mL, 800 IU / mL or about 800 IU / mL, 900 IU / mL or about 900 IU / mL, 1000 IU / mL or about 1000 IU / mL, 1100 IU / mL or about 1100 IU / mL, 1200 IU / mL or about 1200 IU / mL, 1300 IU / mL or about In some embodiments, IL-15 is added to the cell culture medium at a concentration of 1300 IU / mL, 1400 IU / mL or about 1400 IU / mL, 1500 IU / mL or about 1500 IU / mL, 1600 IU / mL or about 1600 IU / mL, 1700 IU / mL or about 1700 IU / mL, 1800 IU / mL or about 1800 IU / mL, 1900 IU / mL or about 1900 IU / mL, or 2000 IU / mL, or any concentration between any of the foregoing. In some embodiments, IL-15 is added to the culture medium at a concentration of 1000 IU / mL or about 1000 IU / mL.

[0197] In some embodiments, the initial expansion (e.g., first expansion) is carried out in the presence of recombinant IL-15 added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 IU / mL or about 1000 IU / mL). In some embodiments, the initial expansion (e.g., first expansion) is carried out in the presence of recombinant IL-15 added at a concentration of 1000 IU / mL or about 1000 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine from IL-2, IL-7, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0198] In some embodiments, recombinant IL-15 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-15 is added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 IU / mL or about 1000 IU / mL) and recombinant IL-2 is added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL). In some embodiments, the initial expansion (e.g., first expansion) is performed in the presence of recombinant IL-15 added at 1000 IU / mL and recombinant IL-2 added at 300 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine from IL-7, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0199] In some embodiments, recombinant IL-7 is added to the culture medium. In some aspects, recombinant IL-7 is added to the culture medium together with one or both of IL-2 and IL-15. In some aspects, recombinant IL-7 and recombinant IL-2 are added to the culture medium. In some aspects, recombinant IL-7 and recombinant IL-15 are added to the culture medium. In some aspects, recombinant IL-7 (e.g., in combination with one or both of IL-2 and IL-15) and one other recombinant regulatory cytokine from IL-23, IL-25, IL-27, or IL-35 are added to the culture medium. IL-7 is a cytokine involved in promoting T cell maintenance and homeostasis. In some cases, IL-7 can enhance the survival and proliferation of memory T cells, particularly the central memory compartment. Recombinant IL-7 is commercially available. In certain embodiments, the recombinant IL-7 is GMP grade (e.g., MACS GMP Recombinant Human IL-7, Miltenyi Biotec).

[0200] Recombinant IL-7 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-7 can be included in the initial T cell expansion (first expansion), e.g., to promote the expansion of TILs to promote their growth, allow their proliferation, and / or stabilize the phenotype from solid tumors. IL-7 can also be included in antigen-presenting cell coculture, as described in Section IB2, e.g., to allow peak activation of neoantigen-reactive T cells prior to their isolation or selection. In some cases, recombinant IL-7 can also be included in the culture to expand tumor-reactive T cells during the second expansion phase, as described in Section IB. The inclusion of recombinant IL-7 in this process can maintain or support the expansion of memory T cell subsets in this process. In some cases, recombinant IL-7 and recombinant IL-15 can be combined in the provided methods to provide activation, survival, and / or expansion of tumor-reactive T cells. In some such embodiments, the combination of recombinant IL-7 and recombinant IL-15 is a substitute for the use of recombinant IL-2 in the culture, and the culture medium does not further contain recombinant IL-2.

[0201] In some embodiments, recombinant IL-7 is administered at a concentration of from 100 IU / mL or about 100 IU / mL, 2000 IU / mL or about 2000 IU / mL, 100 IU / mL or about 100 IU / mL, 1500 IU / mL or about 1500 IU / mL, 100 IU / mL or about 100 IU / mL, 1000 IU / mL or about 1000 IU / mL, 100 IU / mL or about 100 IU / mL, 800 IU / mL or about 800 IU / mL, 100 IU / mL or about 100 IU / mL, 600 IU / mL or about 600 IU / mL, 10 From 0 IU / mL or about 100 IU / mL, 400 IU / mL or about 400 IU / mL, 100 IU / mL or about 100 IU / mL, 200 IU / mL or about 200 IU / mL, 200 IU / mL or about 200 IU / mL, 2000 IU / mL or about 2000 IU / mL, 200 IU / mL or about 200 IU / mL, 1500 IU / mL or about 1500 IU / mL, 200 IU / mL or about 200 IU / mL, 1000 IU / mL or about 1000 IU / mL, 200 IU / mL or about 200 IU / mL, 00IU / mL or about 800IU / mL, 200IU / mL or about 200IU / mL, 600IU / mL or about 600IU / mL, 200IU / mL or about 200IU / mL, 400IU / mL or about 400IU / mL, 400IU / mL or about 400IU / mL, 2000IU / mL or about 2000IU / mL, 400IU / mL or about 400IU / mL, 1500IU / mL or about 1500IU / mL, 400IU / mL or about 400IU / mL, 1000IU / mL or about 1000IU / mL, 4 from 00 IU / mL or about 400 IU / mL, 800 IU / mL or about 800 IU / mL, 400 IU / mL or about 400 IU / mL, from 600 IU / mL or about 600 IU / mL, from 600 IU / mL or about 600 IU / mL, from 2000 IU / mL or about 2000 IU / mL, from 600 IU / mL or about 600 IU / mL, from 1500 IU / mL or about 1500 IU / mL, from 600 IU / mL or about 600 IU / mL, from 1000 IU / mL or about 1000 IU / mL, from 600 IU / mL or about 600 IU / mL,800 IU / mL or about 800 IU / mL, 800 IU / mL or about 800 IU / mL to 2000 IU / mL or about 2000 IU / mL, 800 IU / mL or about 800 IU / mL to 1500 IU / mL or about 1500 IU / mL, 800 IU / mL or about 800 IU / mL to 1000 IU / mL or about 1000 IU / mL In some embodiments, IL-7 is added to the culture medium at a concentration of from 1000 IU / mL or about 1000 IU / mL to 2000 IU / mL or about 2000 IU / mL, from 1000 IU / mL or about 1000 IU / mL to 1500 IU / mL or about 1500 IU / mL, from 1500 IU / mL or about 1500 IU / mL to 2000 IU / mL or about 2000 IU / mL. In some embodiments, IL-7 is added to the culture medium at a concentration of from 1000 IU / mL or about 1000 IU / mL to 2000 IU / mL or about 2000 IU / mL. In some embodiments, IL-7 is added to the culture medium at a concentration of from 600 IU / mL or about 600 IU / mL. In some embodiments, IL-7 is added to the culture medium at a concentration of from 1000 IU / mL or about 1000 IU / mL. ,

[0202] In some embodiments, recombinant IL-7 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-7 is added at a concentration of 400 IU / mL to 2000 IU / mL (e.g., 600 IU / mL or 1000 IU / mL, or about 600 IU / mL or about 1000 IU / mL) and recombinant IL-2 is added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL). In some embodiments, an initial expansion (e.g., a first expansion) is performed in the presence of recombinant IL-7 added at 1000 IU / mL and recombinant IL-2 added at 300 IU / mL. In some embodiments, the first expansion is performed in the presence of recombinant IL-7 added at 600 IU / mL and recombinant IL-2 added at 300 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine from IL-2, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0203] In some embodiments, recombinant IL-15 and IL-7 are added to the culture medium. In some embodiments, recombinant IL-15 is added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 IU / mL or about 1000 IU / mL) and recombinant IL-7 is added at a concentration of 400 IU / mL to 2000 IU / mL (e.g., 600 IU / mL or 1000 IU / mL, or about 600 IU / mL or about 1000 IU / mL). In some embodiments, an initial expansion (e.g., a first expansion) is performed in the presence of recombinant IL-15 added at 1000 IU / mL and recombinant IL-7 added at 1000 IU / mL. In some embodiments, the first expansion is performed in the presence of recombinant IL-15 added at 1000 IU / mL and recombinant IL-7 added at 600 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine from IL-2, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0204] In some embodiments, recombinant IL-21 is added to the culture medium. In some aspects, recombinant IL-21 is added to the culture medium together with one or both of IL-2, IL-7, or IL-15. In some aspects, recombinant IL-21 and recombinant IL-2 are added to the culture medium. In some aspects, recombinant IL-21 and recombinant IL-15 are added to the culture medium. In some aspects, recombinant IL-21 (e.g., in combination with one or more of IL-2, IL-7, and IL-15) and one other recombinant regulatory cytokine from IL-23, IL-25, IL-27, or IL-35 are 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 T cells and CD8 T cells. Recombinant IL-21 is commercially available. In certain embodiments, the recombinant IL-21 is GMP grade (e.g., MACS GMP Recombinant Human IL-21, Miltenyi Biotec).

[0205] Recombinant IL-21 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-21 can be included in the initial T cell expansion (first expansion) to promote the growth of TILs, e.g., from solid tumors, e.g., by stabilizing the activation, function, and / or proliferation of memory T cells. In some aspects, the presence of IL-21 allows for improved recovery of TILs. Recombinant IL-21 can also be included in antigen-presenting cell cocultures, as described in Section IB2, for its ability to stimulate the expression of T cell activation markers, including the expression of activation markers on neoantigen-reactive TILs. 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 IB2, e.g., to support the proliferation and stabilization of the memory phenotype.

[0206] In some embodiments, recombinant IL-21 is administered at a concentration of from 0.5 IU / mL or about 0.5 IU / mL to 20 IU / mL or about 20 IU / mL, from 0.5 IU / mL or about 0.5 IU / mL to 15 IU / mL or about 15 IU / mL, from 0.5 IU / mL or about 0.5 IU / mL to 10 IU / mL or about 10 IU / mL, from 0.5 IU / mL or about 0.5 IU / mL to 5 IU / mL or about 5 IU / mL, from 0.5 IU / mL or about 0.5 IU / mL to 2.5 IU / mL or from about 2.5 IU / mL, 0.5 IU / mL or about 0.5 IU / mL, from 1 IU / mL or about 1 IU / mL, from 20 IU / mL or about 20 IU / mL, from 15 IU / mL or about 15 IU / mL, from 10 IU / mL or about 10 IU / mL, from 5 IU / mL or about 5 IU / mL, from 1 IU / mL or about 1 IU / mL , 2.5 IU / mL or about 2.5 IU / mL, 2.5 IU / mL or about 2.5 IU / mL to 20 IU / mL or about 20 IU / mL, 2.5 IU / mL or about 2.5 IU / mL to 15 IU / mL or about 15 IU / mL, 2.5 IU / mL or about 2.5 IU / mL to 10 IU / mL or about 10 IU / mL, 2.5 IU / mL or about 2.5 IU / mL to 5 IU / mL or about 5 IU / mL, 5 IU / mL or about 5 IU / mL to 20 IU / mL or about 2 In some embodiments, IL-21 is added to the culture medium at a concentration of from 0 IU / mL, 5 IU / mL or about 5 IU / mL to 15 IU / mL or about 15 IU / mL, 5 IU / mL or about 5 IU / mL to 10 IU / mL or about 10 IU / mL, 10 IU / mL or about 10 IU / mL to 20 IU / mL or about 20 IU / mL, 10 IU / mL or about 10 IU / mL to 15 IU / mL or about 15 IU / mL, or 15 IU / mL or about 15 IU / mL to 20 IU / mL or about 20 IU / mL. In some embodiments, IL-21 is added to the culture medium in an amount of from 0.5 IU / mL or about 0.5 IU / mL to 2.5 IU / mL or about 2.5 IU / mL.In some embodiments, IL-21 is added to the culture medium at or about 1 IU / mL.

[0207] In some embodiments, the incubation is performed with an even higher dose of IL-21.

[0208] In some embodiments, the recombinant IL-21 is administered at a concentration of from 500 IU / mL or about 500 IU / mL to 5000 IU / mL or about 5000 IU / mL, e.g., from 500 IU / mL or about 500 IU / mL to 4000 IU / mL or about 4000 IU / mL, from 500 IU / mL or about 500 IU / mL to 2000 IU / mL or about 2000 IU / mL, from 500 IU / mL or about 500 IU / mL to 1500 IU / mL or about 1500 IU / mL, from 500 IU / mL or about 500 IU / mL to 1000 IU / mL. / mL or about 1000 IU / mL, 500 IU / mL or about 500 IU / mL, 750 IU / mL or about 750 IU / mL, 750 IU / mL or about 750 IU / mL, 5000 IU / mL or about 5000 IU / mL, 750 IU / mL or about 750 IU / mL, 4000 IU / mL or about 4000 IU / mL, 750 IU / mL or about 750 IU / mL, 2000 IU / mL or about 2000 IU / mL, 750 IU / mL or about 750 IU / mL, 1500 IU / mL or From about 1500 IU / mL, 750 IU / mL or about 750 IU / mL, 1000 IU / mL or about 1000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 5000 IU / mL or about 5000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 1500 IU / mL or from about 1500 IU / mL, 1500 IU / mL or about 1500 IU / mL, 5000 IU / mL or about 5000 IU / mL, 1500 IU / mL or about 1500 IU / mL, 4000 IU / mL or about 4000 IU / mL, 1500 IU / mL or about 1500 IU / mL, 2000 IU / mL or about 2000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 5000 IU / mL or about 5000 IU / mL, for example, from 2000 IU / mL or about 2000 IU / mL,In some embodiments, recombinant IL-21 is added to the culture medium at a concentration of 500 IU / mL or about 500 IU / mL, 600 IU / mL or about 600 IU / mL, 700 IU / mL or about 700 IU / mL, 800 IU / mL or about 800 IU / mL, 900 IU / mL or about 900 IU / mL, 1000 IU / mL or about 1000 IU / mL, 1100 IU / mL or about 1100 IU / mL, 1200 IU / mL or about 1200 IU / mL, 1300 IU / mL or about In some embodiments, IL-21 is added to the cell culture medium at a concentration of 1300 IU / mL, 1400 IU / mL or about 1400 IU / mL, 1500 IU / mL or about 1500 IU / mL, 1600 IU / mL or about 1600 IU / mL, 1700 IU / mL or about 1700 IU / mL, 1800 IU / mL or about 1800 IU / mL, 1900 IU / mL or about 1900 IU / mL, or 2000 IU / mL, or any concentration between any of the foregoing. In some embodiments, IL-21 is added to the culture medium at a concentration of 1000 IU / mL or about 1000 IU / mL.

[0209] In some embodiments, recombinant IL-21 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-21 is added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 IU / mL or about 1000 IU / mL) and recombinant IL-2 is added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL). In some embodiments, the initial expansion (e.g., first expansion) is performed in the presence of recombinant IL-21 added at 1000 IU / mL and recombinant IL-2 added at 300 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine from IL-7, IL-15, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0210] In some embodiments, recombinant IL-23 is present in the cell culture medium. IL-23 is a cytokine that signals through the IL-23 receptor, which is typically upregulated on activated memory T cells. IL-23 binding leads to activation of the JAK / STAT pathway, i.e., JAK2 and STAT3. JAK signaling leads to activation of NF-kB p50 / p65, which binds to the IL17 promoter and upregulates its expression. STAT3 activation leads to direct binding of the IL-17 promoter and RORyT. In some aspects, this dual mechanism results in strong and sustained IL-17 production for maintenance of the Th17 cell subset. IL-23 plays a role in inflammatory T cell responses and is a target for therapeutic intervention in many autoimmune diseases. In some aspects, IL-23's activity as a pro-inflammatory cytokine, known to act on memory T cells, can be used to activate and expand antigen-experienced T cells.

[0211] IL-23 comprises two subunits linked by a disulfide bond: the P19 (IL23a) subunit and the P40 (IL12b) subunit. An exemplary sequence of human IL-23 is set forth below: TIFF0007817937000001.tif85154.

[0212] In some embodiments, the recombinant IL-23 has at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:1, and and a heterodimer comprising an amino acid sequence having at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:2, wherein both subunits of the heterodimer are linked by a disulfide bond, and the sequence exhibits an activity of recombinant IL-23, e.g., the ability to bind and mediate signaling via the IL-23 receptor. In some embodiments, the recombinant IL-23 has the sequences set forth in SEQ ID NO:1 and SEQ ID NO:2 linked by a disulfide bond. The exemplary SEQ ID NOs should not be construed as limiting. For example, a particular sequence of recombinant IL-23, or individual subunits thereof, can be several amino acids longer or shorter, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids, at either or both the N-terminus or C-terminus, than the amino acid sequence set forth in the respective SEQ ID NOs: 1 and / or 2. In some embodiments, the recombinant IL-23 is a human sequence. In certain embodiments, the IL-23 is a GMP-grade reagent.

[0213] Recombinant IL-23 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-23 can be included in the initial T cell expansion (first expansion), e.g., solid tumor cultures, or other samples known or predicted to contain tumor-reactive T cells or TILs, to promote preferential activation and recovery of antigen-experienced T cells and increase the frequency of neoantigen-reactive cells isolated from bulk T cells. In some cases, recombinant IL-23 can also be included in the culture to expand selected tumor-reactive T cells during the second expansion phase, as described in Section ID, thereby promoting their sustained activity and proliferation during the expansion process.

[0214] In some embodiments, the recombinant IL-23 is administered at a concentration of from 1 nM or about 1 nM to 500 nM or about 500 nM, e.g., from 1 nM or about 1 nM to 400 nM or about 400 nM, from 1 nM or about 1 nM to 300 nM or about 300 nM, from 1 nM or about 1 nM to 200 nM or about 200 nM, from 1 nM or about 1 nM to 100 nM or about 100 nM, from 1 nM or about 1 nM to 50 nM or about 50 nM, from 1 nM or about 1 nM to 25 nM or about 25 nM, from 1 nM or about 1 nM to 10 nM, or from about 10 nM, 1 nM or about 1 nM, 5 nM or about 5 nM, 5 nM or about 5 nM, 500 nM or about 500 nM, 5 nM or about 5 nM, 400 nM or about 400 nM, 5 nM or about 5 nM, 300 nM or about 300 nM, 5 nM or about 5 nM, 200 nM or about 200 nM, 5 nM or about 5 nM, 100 nM or about 100 nM, 5 nM or about 5 nM, 50 nM or about 50 nM, 5 nM or about 5 nM, 25 nM or about 25 nM, 5 nM or From about 5 nM, 10 nM or about 10 nM, 10 nM or about 10 nM to 500 nM or about 500 nM, 10 nM or about 10 nM to 400 nM or about 400 nM, 10 nM or about 10 nM to 300 nM or about 300 nM, 10 nM or about 10 nM to 200 nM or about 200 nM, 10 nM or about 10 nM to 100 nM or about 100 nM, 10 nM or about 10 nM to 50 nM or about 50 nM, 10 nM or about 10 nM to 25 nM or about 25 nM, 25 nM or or about 25 nM to 500 nM, 25 nM or about 25 nM to 400 nM or about 400 nM, 25 nM or about 25 nM to 300 nM or about 300 nM, 25 nM or about 25 nM to 200 nM or about 200 nM, 25 nM or about 25 nM to 100 nM or about 100 nM, 25 nM or about 25 nM to 50 nM or about 50 nM, 50 nM or about 50 nM to 500 nM, 50 nM or about 50 nM to 400 nM or about 400 nM,From 50 nM or about 50 nM, 300 nM or about 300 nM, 50 nM or about 50 nM to 200 nM or about 200 nM, 50 nM or about 50 nM to 100 nM or about 100 nM, 100 nM or about 100 nM to 500 nM or about 500 nM, 100 nM or about 100 nM to 400 nM or about 400 nM, 100 nM or about 100 nM to 300 nM or about 300 nM, 100 nM or about 100 nM to 200 nM or about The compound is added to the culture medium at a concentration of from 200 nM, 200 nM or about 200 nM, 500 nM or about 500 nM, 200 nM or about 200 nM, 400 nM or about 400 nM, 200 nM or about 200 nM, 300 nM or about 300 nM, 300 nM or about 300 nM, 500 nM or about 500 nM, 300 nM or about 300 nM, 400 nM or about 400 nM, or 400 nM or about 400 nM, 500 nM or about 500 nM. In some embodiments, recombinant IL-23 is added to the culture medium at a concentration of 5 nM or about 5 nM, 10 nM or about 10 nM, 20 nM or about 20 nM, 30 nM or about 30 nM, 40 nM or about 40 nM, 50 nM or about 50 nM, 60 nM or about 60 nM, 70 nM or about 70 nM, 80 nM or about 80 nM, 90 nM or about 90 nM, or 100 nM or about 100 nM, or any value between any of the foregoing.

[0215] In some embodiments, recombinant IL-23 is added to the culture medium at a concentration of from at or about 0.1 ng / mL to at or about 2000 ng / mL. In some embodiments, the recombinant IL-23 is at a concentration of from 0.1 ng / mL or about 0.1 ng / mL to 1000 ng / mL or about 1000 ng / mL, e.g., from 0.1 ng / mL or about 0.1 ng / mL to 500 ng / mL or about 500 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 250 ng / mL or about 250 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 100 ng / mL or about 100 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 50 ng / mL or about 50 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 10 ng / mL or about 10 ng / mL, from 0.1 ng / mL or about 0.From 1ng / mL, 1ng / mL or about 1ng / mL, 1ng / mL or about 1ng / mL to 1000ng / mL or about 1000ng / mL, 1ng / mL or about 1ng / mL to 500ng / mL or about 500ng / mL, 1ng / mL or about 1ng / mL to 250ng / mL or about 250ng / mL, 1ng / mL or about 1ng / mL to 100ng / mL or about 100ng / mL, 1ng / mL or about 1ng / mL to 50ng / mL or about 50ng / mL, 1ng / mL or from about 1 ng / mL, 10 ng / mL or about 10 ng / mL, 10 ng / mL or about 10 ng / mL, 1000 ng / mL or about 1000 ng / mL, 10 ng / mL or about 10 ng / mL, 500 ng / mL or about 500 ng / mL, 10 ng / mL or about 10 ng / mL, 250 ng / mL or about 250 ng / mL, 10 ng / mL or about 10 ng / mL, 100 ng / mL or about 100 ng / mL, 10 ng / mL or about 10 ng / mL, 50 ng / mL or is from about 50 ng / mL, 50 ng / mL or about 50 ng / mL, 1000 ng / mL or about 1000 ng / mL, 50 ng / mL or about 50 ng / mL, 500 ng / mL or about 500 ng / mL, 50 ng / mL or about 50 ng / mL, 250 ng / mL or about 250 ng / mL, 50 ng / mL or about 50 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 1000 ng / mL, The antibody is added to the culture medium at a concentration of from about 100 ng / mL to about 500 ng / mL, from about 100 ng / mL to about 250 ng / mL, from about 250 ng / mL to about 250 ng / mL, from about 1000 ng / mL to about 1000 ng / mL, from about 250 ng / mL to about 500 ng / mL, or from about 500 ng / mL to about 1000 ng / mL.

[0216] In some embodiments, recombinant IL-23 is added to the culture medium at a concentration of at or about 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or any value between any of the foregoing.

[0217] In some embodiments, the recombinant IL-23 is at or about 200ng / mL, 300ng / mL or about 300ng / mL, 400ng / mL or about 400ng / mL, 500ng / mL or about 500ng / mL, 600ng / mL or about 600ng / mL, 700ng / mL or about 700ng / mL, 800ng / mL or about 800ng / mL, 900ng / mL or about 900ng / mL or about 1000ng / mL, 1200ng / mL or about 1200ng / mL, 1400ng / mL or about 1400ng / mL, 1600ng / mL or about 1600ng / mL, 1800ng / mL or about 1800ng / mL, or 2000ng / mL or about 2000ng / mL, or any value between any of the foregoing.

[0218] In some embodiments, recombinant IL-2 and recombinant IL-23 are added to the culture medium. In some embodiments, recombinant IL-2 is added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL), and recombinant IL-23 is added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, the initial expansion is carried out in the presence of recombinant IL-2 added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL) and recombinant IL-23 added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, at least one other recombinant regulatory cytokine from IL-7, IL-21, IL-15, IL-25, IL-27, or IL-35 is added to the culture medium.

[0219] In some embodiments, recombinant IL-25 is present in the cell culture medium. IL-25 belongs to the IL-17 family and is also known as IL-17E. IL-25 binds to a heterodimeric receptor composed of two subunits, IL-17RA and IL-17RB. IL-25 is a pro-inflammatory cytokine that typically supports the development of Th2 cells. IL-25 inhibits IFN-mediated signaling. γ It has been shown to decrease the production of IL-25 and bias the immune response away from Th1 / Th17 responses. IL-25 has also been shown to stimulate NFkB activity, which can broadly activate cells.

[0220] An exemplary sequence for human IL-25 is set forth below: TIFF0007817937000002.tif25154.

[0221] In some embodiments, the recombinant IL-25 has an amino acid sequence having at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:3, and the sequence exhibits an activity of the recombinant IL-25, e.g., the ability to bind to its heterodimeric receptor subunits and mediate signaling through the IL-25 (IL-17RA / IL-17RB) receptor. In some embodiments, the recombinant IL-25 has the sequence set forth in SEQ ID NO:3. The exemplary SEQ ID NOs should not be construed as limiting. For example, a particular sequence of recombinant IL-25, or individual subunits thereof, can be several amino acids longer or shorter, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids, at either or both the N-terminus or C-terminus, than the amino acid sequence set forth in the respective SEQ ID NO:3. In some embodiments, the recombinant IL-25 is a human sequence. In certain embodiments, the IL-25 is a GMP-grade reagent.

[0222] In some embodiments, recombinant IL-25 is administered at a concentration of from at or about 0.001 nM to at or about 10 nM, e.g., from 0.001 nM or about 0.001 nM to 5 nM or about 5 nM, from 0.001 nM or about 0.001 nM to 2.5 nM or about 2.5 nM, from 0.001 nM or about 0.001 nM to 1 nM or about 1 nM, from 0.001 nM or about 0.001 nM to 0.5 nM or about 0.5 nM, from 0.001 nM or about 0.001 nM to 0.1 nM or about 0.1 nM, nM or about 0.001 nM to 0.05 nM or about 0.05 nM, 0.001 nM or about 0.001 nM to 0.01 nM or about 0.01 nM, 0.001 nM or about 0.001 nM to 0.005 nM or about 0.005 nM, 0.005 nM or about 0.005 nM to 10 nM or about 10 nM, 0.005 nM or about 0.005 nM to 5 nM or about 5 nM, 0.005 nM or about 0.005 nM to 2.5 nM or about 2.5 nM, 0.005 nM or about 0.005 nM to 1 nM M or about 1 nM, 0.005 nM or about 0.005 nM to 0.5 nM or about 0.5 nM, 0.005 nM or about 0.005 nM to 0.1 nM or about 0.1 nM, 0.005 nM or about 0.005 nM to 0.05 nM or about 0.05 nM, 0.005 nM or about 0.005 nM to 0.01 nM or about 0.01 nM, 0.01 nM or about 0.01 nM to 10 nM, 0.01 nM or about 0.01 nM to 5 nM or about 5 nM, 0.01 nM or about 0.01 nM from 2.5 nM or about 2.5 nM, from 0.01 nM or about 0.01 nM, from 1 nM or about 1 nM, from 0.01 nM or about 0.01 nM, from 0.5 nM or about 0.5 nM, from 0.01 nM or about 0.01 nM, from 0.1 nM or about 0.1 nM, from 0.01 nM or about 0.01 nM, from 0.05 nM or about 0.05 nM, from 0.05 nM or about 0.05 nM, from 10 nM or about 10 nM, from 0.05 nM or about 0.05 nM, from 5 nM or about 5 nM, from 0.05 nM or about 0.05 nM,5 nM or about 2.5 nM, from 0.05 nM or about 0.05 nM, 1 nM or about 1 nM, from 0.05 nM or about 0.05 nM, from 0.5 nM or about 0.5 nM, from 0.05 nM or about 0.05 nM, from 0.1 nM or about 0.1 nM, from 0.1 nM or about 0.1 nM, from 10 nM or about 10 nM, from 0.1 nM or about 0.1 nM, from 5 nM or about 5 nM, from 0.1 nM or about 0.1 nM, from 2.5 nM or about 2.5 nM, from 0.1 nM or about 0.1 nM, from 1 nM or about 1 nM, from 0.1 nM or about 0.1 nM, from 0.5 nM or about 0.5 nM, from 0.5 nM or about 0.5 nM, or about 10 nM, from 0.5 nM or about 0.5 nM, from 5 nM or about 5 nM, from 0.5 nM or about 0.5 nM, from 2.5 nM or about 2.5 nM, from 0.5 nM or about 0.5 nM, from 1 nM or about 1 nM, from 1 nM or about 1 nM, from 10 nM or about 10 nM, from 1 nM or about 1 nM, from 5 nM or about 5 nM, from 1 nM or about 1 nM, from 2.5 nM or about 2.5 nM, from 2.5 nM or about 2.5 nM, from 10 nM or about 10 nM, from 2.5 nM or about 2.5 nM, from 5 nM or about 5 nM, or from 5 nM or about 5 nM, from 10 nM or about 10 nM. In some embodiments, recombinant IL-25 is added to the culture medium at a concentration of 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, or 1 nM, 1.5 nM, or 2 nM, or about 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, or 1 nM, 1.5 nM, or 2 nM, or any value between any of the foregoing.

[0223] In some embodiments, the recombinant IL-25 is present in a concentration of from 0.01 ng / mL or about 0.01 ng / mL to 500 ng / mL or about 500 ng / mL, 0.01 ng / mL or about 0.01 ng / mL to 250 ng / mL or about 250 ng / mL, 0.01 ng / mL or about 0.01 ng / mL to 100 ng / mL or about 100 ng / mL, 0.01 ng / mL or about 0.01 ng / mL to 50 ng / mL or about 50 ng / mL, 0.01 ng / mL or about 0.01 ng / mL to 20 ng / mL or about 2 From 0 ng / mL, 0.01 ng / mL or about 0.01 ng / mL, 10 ng / mL or about 10 ng / mL, 0.01 ng / mL or about 0.01 ng / mL, 5 ng / mL or about 5 ng / mL, 0.01 ng / mL or about 0.01 ng / mL, 1 ng / mL or about 1 ng / mL, 0.01 ng / mL or about 0.01 ng / mL, 0.05 ng / mL or about 0.05 ng / mL, 0.05 ng / mL or about 0.05 ng / mL, 500 ng / mL or about 500 ng / mL, 0.05 ng / mL or about 0.05ng / mL, 250ng / mL or about 250ng / mL, 0.05ng / mL or about 0.05ng / mL, 100ng / mL or about 100ng / mL, 0.05ng / mL or about 0.05ng / mL, 50ng / mL or about 50ng / mL, 0.05ng / mL or about 0.05ng / mL, 20ng / mL or about 20ng / mL, 0.05ng / mL or about 0.05ng / mL, 10ng / mL or about 10ng / mL, 0.05ng / mL or about 0.05ng / mL , from 5ng / mL or about 5ng / mL, 0.05ng / mL or about 0.05ng / mL, from 1ng / mL or about 1ng / mL, from 1ng / mL or about 1ng / mL, from 500ng / mL or about 500ng / mL, from 1ng / mL or about 1ng / mL, from 250ng / mL or about 250ng / mL, from 1ng / mL or about 1ng / mL, from 100ng / mL or about 100ng / mL, from 1ng / mL or about 1ng / mL, from 50ng / mL or about 50ng / mL, from 1ng / mL or about 1ng / mL,From 20ng / mL or about 20ng / mL, from 1ng / mL or about 1ng / mL, from 10ng / mL or about 10ng / mL, from 1ng / mL or about 1ng / mL, from 5ng / mL or about 5ng / mL, from 5ng / mL or about 5ng / mL, from 500ng / mL or about 500ng / mL, from 5ng / mL or about 5ng / mL, from 250ng / mL or about 250ng / mL, from 5ng / mL or about 5ng / mL, from 100ng / mL or about 100ng / mL, from 5ng / mL or about 5ng / mL, ng / mL or about 50 ng / mL, 5 ng / mL or about 5 ng / mL, 20 ng / mL or about 20 ng / mL, 5 ng / mL or about 5 ng / mL, 10 ng / mL or about 10 ng / mL, 10 ng / mL or about 10 ng / mL, 500 ng / mL or about 500 ng / mL, 10 ng / mL or about 10 ng / mL, 250 ng / mL or about 250 ng / mL, 10 ng / mL or about 10 ng / mL, 100 ng / mL or about 100 ng / mL, 10 ng / mL or about 10 ng / mL to 50ng / mL or about 50ng / mL, 10ng / mL or about 10ng / mL to 20ng / mL or about 20ng / mL, 20ng / mL or about 20ng / mL to 500ng / mL or about 500ng / mL, 20ng / mL or about 20ng / mL to 250ng / mL or about 250ng / mL, 20ng / mL or about 20ng / mL to 100ng / mL or about 100ng / mL, 20ng / mL or about 20ng / mL to 50ng / mL or about 50ng / mL, 50ng from 500ng / mL or about 500ng / mL, 50ng / mL or about 50ng / mL, 250ng / mL or about 250ng / mL, 50ng / mL or about 50ng / mL, 100ng / mL or about 100ng / mL, 100ng / mL or about 100ng / mL, 500ng / mL or about 500ng / mL, 100ng / mL or about 100ng / mL, 250ng / mL or about 250ng / mL, or 250ng / mL or about 250ng / mL,In some embodiments, recombinant IL-25 is added to the culture medium at a concentration of 500 ng / mL or about 500 ng / mL. In some embodiments, recombinant IL-25 is added to the culture medium at a concentration of 1 ng / mL or about 1 ng / mL, 2 ng / mL or about 2 ng / mL, 3 ng / mL or about 3 ng / mL, 4 ng / mL or about 4 ng / mL, 5 ng / mL or about 5 ng / mL, 6 ng / mL or about 6 ng / mL, 7 ng / mL or about 7 ng / mL, 8 ng / mL or about 8 ng / mL, 9 ng / mL or about 9 ng / mL, 10 ng / mL or about 10 ng / mL, 15 ng / mL or about 15 ng / mL, or 20 ng / mL or about 20 ng / mL, or any value between any of the foregoing.

[0224] Recombinant IL-25 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-25 can be included in the initial T cell expansion (first expansion), for example, during the isolation and expansion of TILs from solid tissue, to aid in the preservation and expansion of Th2 CD4 T cells. In some cases, recombinant IL-25 can also be included in the culture to expand selected tumor-reactive T cells during the second expansion phase, as described in Section ID. For example, IL-25 can be included in the culture medium on days 9-16 during TIL expansion to promote CD4 / CD8 balance and / or maintain T cell activation rates. The use of IL-25 can promote T cell proliferation as well as promote NFkB activity, which can help enhance T cell expansion and activation.

[0225] In some embodiments, the recombinant IL-25 is at a concentration of from 0.1 ng / mL or about 0.1 ng / mL to 2000 ng / mL or about 2000 ng / mL, e.g., from 0.1 ng / mL or about 0.1 ng / mL to 1000 ng / mL or about 1000 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 500 ng / mL or about 500 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 250 ng / mL or about 250 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 100 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 50 ng / mL or about 50 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 1 ...From 1ng / mL, 1ng / mL or about 1ng / mL, 1ng / mL or about 1ng / mL to 1000ng / mL or about 1000ng / mL, 1ng / mL or about 1ng / mL to 500ng / mL or about 500ng / mL, 1ng / mL or about 1ng / mL to 250ng / mL or about 250ng / mL, 1ng / mL or about 1ng / mL to 100ng / mL or about 100ng / mL, 1ng / mL or about 1ng / mL to 50ng / mL or about 50ng / mL, 1ng / mL or from about 1 ng / mL, 10 ng / mL or about 10 ng / mL, 10 ng / mL or about 10 ng / mL, 1000 ng / mL or about 1000 ng / mL, 10 ng / mL or about 10 ng / mL, 500 ng / mL or about 500 ng / mL, 10 ng / mL or about 10 ng / mL, 250 ng / mL or about 250 ng / mL, 10 ng / mL or about 10 ng / mL, 100 ng / mL or about 100 ng / mL, 10 ng / mL or about 10 ng / mL, 50 ng / mL or is from about 50 ng / mL, 50 ng / mL or about 50 ng / mL, 1000 ng / mL or about 1000 ng / mL, 50 ng / mL or about 50 ng / mL, 500 ng / mL or about 500 ng / mL, 50 ng / mL or about 50 ng / mL, 250 ng / mL or about 250 ng / mL, 50 ng / mL or about 50 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 1000 ng / mL, The antibody is added to the culture medium at a concentration of from about 100 ng / mL to about 500 ng / mL, from about 100 ng / mL to about 250 ng / mL, from about 250 ng / mL to about 250 ng / mL, from about 1000 ng / mL to about 1000 ng / mL, from about 250 ng / mL to about 500 ng / mL, or from about 500 ng / mL to about 1000 ng / mL.

[0226] In some embodiments, recombinant IL-25 is added to the culture medium at a concentration of at or about 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or any value between any of the foregoing.

[0227] In some embodiments, recombinant IL-25 is at or about 200ng / mL, 300ng / mL or about 300ng / mL, 400ng / mL or about 400ng / mL, 500ng / mL or about 500ng / mL, 600ng / mL or about 600ng / mL, 700ng / mL or about 700ng / mL, 800ng / mL or about 800ng / mL, 900ng / mL or about 900ng / mL or about 1000ng / mL, 1200ng / mL or about 1200ng / mL, 1400ng / mL or about 1400ng / mL, 1600ng / mL or about 1600ng / mL, 1800ng / mL or about 1800ng / mL, or 2000ng / mL or about 2000ng / mL, or any value between any of the foregoing.

[0228] In some embodiments, recombinant IL-2 and recombinant IL-25 are added to the culture medium. In some embodiments, recombinant IL-2 is added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL), and recombinant IL-25 is added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, the initial expansion is carried out in the presence of recombinant IL-2 added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL) and recombinant IL-25 added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, at least one other recombinant regulatory cytokine from IL-7, IL-21, IL-15, IL-23, IL-27, or IL-35 is added to the culture medium.

[0229] In some embodiments, recombinant IL-27 is present in the cell culture medium. IL-27 is a cytokine that signals through the IL-27 receptor, initiating activation of signal transduction pathways including JAK-STAT and p38 MAPK. In some cases, IL-27 can induce or suppress other T cell subsets, such as Tregs and, in some cases, TH1 cells. IL-27 can regulate Treg responses and program effector T cells into stem-like memory effector cells, which can enhance T cell survival within the tumor microenvironment.

[0230] IL-27 is a heterodimer of two chains, IL27A (IL27p28) and IL27B (EBI3). An exemplary sequence of human IL-27 is set forth below: TIFF0007817937000003.tif85148.

[0231] In some embodiments, the recombinant IL-27 has at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:4, and and a heterodimer comprising an amino acid sequence having at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:5, wherein the heterodimer exhibits an activity of recombinant IL-27, e.g., the ability to bind and mediate signaling via the IL-27 receptor. In some embodiments, the recombinant IL-27 has the sequences set forth in SEQ ID NO:4 and SEQ ID NO:5 linked as a heterodimer. The exemplary SEQ ID NOs should not be construed as limiting. For example, a particular sequence of recombinant IL-27, or individual subunits thereof, can be a few amino acids longer or shorter, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids, at either or both the N-terminus or C-terminus, than the amino acid sequence set forth in the respective SEQ ID NOs:4 and / or 5. In some embodiments, the recombinant IL-27 is a human sequence. In certain embodiments, the IL-27 is a GMP-grade reagent.

[0232] Recombinant IL-27 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-27 can be included in the initial T cell expansion (first expansion), e.g., solid tumor cultures, or other samples known or predicted to contain tumor-reactive T cells or TILs, to promote preferential activation and recovery of antigen-experienced T cells and increase the frequency of neoantigen-reactive cells isolated from bulk T cells. In some cases, recombinant IL-27 can also be included in the culture to expand selected tumor-reactive T cells during the final expansion (e.g., second expansion) phase, as described in Section IE, thereby promoting their sustained activity and proliferation during the expansion process.

[0233] In some embodiments, the recombinant IL-27 is at a concentration of from 0.1 ng / mL or about 0.1 ng / mL to 2000 ng / mL or about 2000 ng / mL, e.g., from 0.1 ng / mL or about 0.1 ng / mL to 1000 ng / mL or about 1000 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 500 ng / mL or about 500 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 250 ng / mL or about 250 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 100 ng / mL or about From 100ng / mL, 0.1ng / mL or about 0.1ng / mL, from 50ng / mL or about 50ng / mL, from 0.1ng / mL or about 0.1ng / mL, from 10ng / mL or about 10ng / mL, from 0.1ng / mL or about 0.1ng / mL, from 1ng / mL or about 1ng / mL, from 1ng / mL or about 1ng / mL, from 1000ng / mL or about 1000ng / mL, from 1ng / mL or about 1ng / mL, from 500ng / mL or about 500ng / mL, from 1ng / mL or about 1ng / mL, ng / mL or about 250 ng / mL, 1 ng / mL or about 1 ng / mL, 100 ng / mL or about 100 ng / mL, 1 ng / mL or about 1 ng / mL, 50 ng / mL or about 50 ng / mL, 1 ng / mL or about 1 ng / mL, 10 ng / mL or about 10 ng / mL, 10 ng / mL or about 10 ng / mL, 1000 ng / mL or about 1000 ng / mL, 10 ng / mL or about 10 ng / mL, 500 ng / mL or about 500 ng / mL, 10 ng / mL or about 10 ng / mL / mL to 250ng / mL or about 250ng / mL, 10ng / mL or about 10ng / mL to 100ng / mL or about 100ng / mL, 10ng / mL or about 10ng / mL to 50ng / mL or about 50ng / mL, 50ng / mL or about 50ng / mL to 1000ng / mL or about 1000ng / mL, 50ng / mL or about 50ng / mL to 500ng / mL or about 500ng / mL, 50ng / mL or about 50ng / mL to 250ng / mL or about 250ng / mL,It is added to the culture medium at a concentration of from 50 ng / mL or about 50 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 100 ng / mL, 1000 ng / mL or about 1000 ng / mL, 100 ng / mL or about 100 ng / mL, 500 ng / mL or about 500 ng / mL, 100 ng / mL or about 100 ng / mL, 250 ng / mL or about 250 ng / mL, 250 ng / mL or about 250 ng / mL, 1000 ng / mL or about 1000 ng / mL, 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 500 ng / mL or about 500 ng / mL, 1000 ng / mL or about 1000 ng / mL. In some embodiments, the concentration is between 400 ng / mL and 500 ng / mL.

[0234] In some embodiments, recombinant IL-27 is at or about 200ng / mL, 300ng / mL or about 300ng / mL, 400ng / mL or about 400ng / mL, 500ng / mL or about 500ng / mL, 600ng / mL or about 600ng / mL, 700ng / mL or about 700ng / mL, 800ng / mL or about 800ng / mL, 900ng / mL or about 900ng / mL or about 1000ng / mL, 1200ng / mL or about 1200ng / mL, 1400ng / mL or about 1400ng / mL, 1600ng / mL or about 1600ng / mL, 1800ng / mL or about 1800ng / mL, or 2000ng / mL or about 2000ng / mL, or any value between any of the foregoing.

[0235] In some embodiments, recombinant IL-2 and recombinant IL-27 are added to the culture medium. In some embodiments, recombinant IL-2 is added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL), and recombinant IL-27 is added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, the initial expansion is carried out in the presence of recombinant IL-2 added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL) and recombinant IL-27 added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, at least one other recombinant regulatory cytokine from IL-7, IL-21, IL-15, IL-23, IL-25, or IL-35 is added to the culture medium.

[0236] In some embodiments, recombinant IL-35 is present in the cell culture medium. IL-35 is a cytokine that can suppress inflammatory responses in some cases. IL-35 also has selective activity on various T cell subsets. In T cells, IL-35 binds to gp130 and IL-12Rβ2 and signals via either the gp130 / IL-12Rβ2 heterodimer or homodimer of each subunit. Engagement of the receptor by IL-35 leads to STAT activation and signal transduction, for example, via the JAK-STAT-mediated pathway.

[0237] IL-35 is a heterodimeric protein containing the p35 subunit (IL-12α) derived from IL-12 and the β subunit (EBI3) derived from IL-27. TIFF0007817937000004.tif85146

[0238] In some embodiments, the recombinant IL-35 has at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:6, and and a heterodimer comprising an amino acid sequence having at least, or at least about, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence set forth in SEQ ID NO:5, wherein the heterodimer exhibits an activity of recombinant IL-35, e.g., the ability to bind to and mediate signaling via an IL-35 receptor (e.g., gp130 and IL-12Rβ2 subunit). In some embodiments, the recombinant IL-35 has the sequences set forth in SEQ ID NO:6 and SEQ ID NO:5 linked as a heterodimer. The exemplary SEQ ID NOs should not be construed as limiting. For example, a particular sequence of recombinant IL-35, or individual subunits thereof, can be several amino acids longer or shorter, e.g., 1 to 10, e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids, at either or both the N-terminus or C-terminus, than the amino acid sequence set forth in the respective SEQ ID NOs: 4 and / or 5. In some embodiments, the recombinant IL-35 is a human sequence. In certain embodiments, the IL-35 is a GMP-grade reagent.

[0239] Recombinant IL-35 can be included in the cell culture medium during various stages of the provided processes. In some cases, recombinant IL-35 can be included in the initial T cell expansion (first expansion), e.g., solid tumor cultures, or other samples known or predicted to contain tumor-reactive T cells or TILs, to promote preferential activation and recovery of antigen-experienced T cells and increase the frequency of neoantigen-reactive cells isolated from bulk T cells. In some cases, recombinant IL-35 can also be included in the culture to expand selected tumor-reactive T cells during the final expansion (e.g., second expansion) phase, as described in Section ID, thereby promoting their sustained activity and proliferation during the expansion process.

[0240] In some embodiments, recombinant IL-35 is present at a concentration of from 0.1 ng / mL or about 0.1 ng / mL to 2000 ng / mL or about 2000 ng / mL, e.g., from 0.1 ng / mL or about 0.1 ng / mL to 1000 ng / mL or about 1000 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 500 ng / mL or about 500 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 250 ng / mL or about 250 ng / mL, from 0.1 ng / mL or about 0.1 ng / mL to 100 ng / mL or about From 100ng / mL, 0.1ng / mL or about 0.1ng / mL, from 50ng / mL or about 50ng / mL, from 0.1ng / mL or about 0.1ng / mL, from 10ng / mL or about 10ng / mL, from 0.1ng / mL or about 0.1ng / mL, from 1ng / mL or about 1ng / mL, from 1ng / mL or about 1ng / mL, from 1000ng / mL or about 1000ng / mL, from 1ng / mL or about 1ng / mL, from 500ng / mL or about 500ng / mL, from 1ng / mL or about 1ng / mL, ng / mL or about 250 ng / mL, 1 ng / mL or about 1 ng / mL, 100 ng / mL or about 100 ng / mL, 1 ng / mL or about 1 ng / mL, 50 ng / mL or about 50 ng / mL, 1 ng / mL or about 1 ng / mL, 10 ng / mL or about 10 ng / mL, 10 ng / mL or about 10 ng / mL, 1000 ng / mL or about 1000 ng / mL, 10 ng / mL or about 10 ng / mL, 500 ng / mL or about 500 ng / mL, 10 ng / mL or about 10 ng / mL / mL to 250ng / mL or about 250ng / mL, 10ng / mL or about 10ng / mL to 100ng / mL or about 100ng / mL, 10ng / mL or about 10ng / mL to 50ng / mL or about 50ng / mL, 50ng / mL or about 50ng / mL to 1000ng / mL or about 1000ng / mL, 50ng / mL or about 50ng / mL to 500ng / mL or about 500ng / mL, 50ng / mL or about 50ng / mL to 250ng / mL or about 250ng / mL,It is added to the culture medium at a concentration of from 50 ng / mL or about 50 ng / mL, 100 ng / mL or about 100 ng / mL, 100 ng / mL or about 100 ng / mL, 1000 ng / mL or about 1000 ng / mL, 100 ng / mL or about 100 ng / mL, 500 ng / mL or about 500 ng / mL, 100 ng / mL or about 100 ng / mL, 250 ng / mL or about 250 ng / mL, 250 ng / mL or about 250 ng / mL, 1000 ng / mL or about 1000 ng / mL, 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 500 ng / mL or about 500 ng / mL, 1000 ng / mL or about 1000 ng / mL. In some embodiments, the concentration is between 400 ng / mL and 500 ng / mL.

[0241] In some embodiments, recombinant IL-35 is at or about 200ng / mL, 300ng / mL or about 300ng / mL, 400ng / mL or about 400ng / mL, 500ng / mL or about 500ng / mL, 600ng / mL or about 600ng / mL, 700ng / mL or about 700ng / mL, 800ng / mL or about 800ng / mL, 900ng / mL or about 900ng / mL or about 1000ng / mL, 1200ng / mL or about 1200ng / mL, 1400ng / mL or about 1400ng / mL, 1600ng / mL or about 1600ng / mL, 1800ng / mL or about 1800ng / mL, or 2000ng / mL or about 2000ng / mL, or any value between any of the foregoing.

[0242] In some embodiments, recombinant IL-2 and recombinant IL-35 are added to the culture medium. In some embodiments, recombinant IL-2 is added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL), and recombinant IL-35 is added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, the initial expansion is carried out in the presence of recombinant IL-2 added at a concentration of 200 IU / mL to 1000 IU / mL (e.g., 300 IU / mL or about 300 IU / mL) and recombinant IL-35 added at a concentration of 100 ng / mL to 2000 ng / mL (e.g., 250 ng / mL or about 250 ng / mL to 1000 ng / mL, e.g., 250 ng / mL or about 250 ng / mL, 500 ng / mL or about 500 ng / mL, or 1000 ng / mL or about 1000 ng / mL). In some embodiments, at least one other recombinant regulatory cytokine from IL-7, IL-21, IL-15, IL-23, IL-25, or IL-27 is added to the culture medium.

[0243] In certain embodiments, for example, the T cell stimulator present during incubation for cell expansion contains recombinant IL-2. In some embodiments, one or more other stimulatory substances may be included, for example, one or more other recombinant cytokines from IL-7, IL-15, IL-21, IL-25, and / or IL-23, or an anti-CD3 antibody (e.g., OKT-3). In such cases of anti-CD3 antibodies (e.g., OKT-3), the T cell stimulatory substance may also include a costimulatory substance, such as provided by antigen-presenting feeder cells, such as PBMCs, or a soluble anti-CD28 antibody.

[0244] In certain embodiments, for example, the T cell stimulatory agents present during the incubation for cell expansion include recombinant IL-2 and anti-CD3 antibodies.

[0245] In certain embodiments, for example, the T cell stimulants present during the incubation for cell expansion include recombinant IL-2, an anti-CD3 antibody, e.g., OKT-3, and antigen-presenting feeder cells, e.g., PBMCs.

[0246] In certain embodiments, for example, the T cell stimuli present during incubation for cell expansion include 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 the anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and the anti-CD28 antibody are bound to a solid surface, such as beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0247] In certain embodiments, for example, the T cell stimulants present during the incubation for cell expansion include recombinant IL-2, recombinant IL-15, recombinant IL-7, an anti-CD3 antibody, e.g., OKT-3, and antigen-presenting feeder cells, e.g., PBMCs.

[0248] In certain embodiments, for example, the T cell stimuli present during incubation for cell expansion include 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 the anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and the anti-CD28 antibody are bound to a solid surface, such as beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0249] In certain embodiments, for example, the T cell stimulants present during the incubation for cell expansion include recombinant IL-15 and recombinant IL-7, an anti-CD3 antibody, e.g., OKT-3, and antigen-presenting feeder cells, e.g., PBMCs.

[0250] In certain embodiments, for example, T cell stimulants present during incubation for cell expansion include 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 the anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and the anti-CD28 antibody are bound to a solid surface, such as beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0251] In some embodiments, incubation with the T cell stimulator 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% CO2. The culture medium containing the T cell stimulator can be serum-free medium.

[0252] In some embodiments, incubation with the T cell stimulator is carried out for 1 day or about 1 day, e.g., generally 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days, or for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days, or any range of time between any of the foregoing. In some embodiments, incubation with the T cell stimulator is carried out for 7 to 21 days, e.g., 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, or 21 days, or any range of time between any of the foregoing. In some embodiments, incubation is carried out for 7 to 14 days. In some embodiments, incubation is carried out for 7 to 10 days. In some embodiments, the incubation is for 7 days or about 7 days. In some embodiments, the incubation is for 8 days or about 8 days. In some embodiments, the incubation is for 9 days or about 9 days. In some embodiments, the incubation is for 10 days or about 10 days.

[0253] In some embodiments, incubation with the T cell stimulator is a minimal expansion so as not to result in downregulation of T cell activation markers (e.g., PD-1, CD39, and / or TIGIT). For example, incubation with the T cell stimulator in initial expansion is a short-term culture such that the markers CD39, PD1, and / or TIGIT are still present during the sorting process and the cells do not downregulate those markers.

[0254] In some embodiments, incubation with a T cell stimulator, e.g., for the initial expansion of T cells in the input sample, is carried out for 1 day or about 1 day. In some embodiments, incubation with a T cell stimulator, e.g., for the initial expansion of T cells in the input sample, is carried out for 2 days or about 2 days. In some embodiments, incubation with a T cell stimulator, e.g., for the initial expansion of T cells in the input sample, is carried out for 3 days or about 3 days. In some embodiments, incubation with a T cell stimulator, e.g., for the initial expansion of T cells in the input sample, is carried out for 4 days or about 4 days. In some embodiments, incubation with a T cell stimulator, e.g., for the initial expansion of T cells in the input sample, is carried out for 5 days or about 5 days. In some embodiments, incubation with a T cell stimulator, e.g., for the initial expansion of T cells in the input sample, is carried out for 6 days or about 6 days. In some embodiments, incubation with a T cell stimulator, for example for the initial expansion of T cells in the input sample, is carried out for 7 days or about 7 days.

[0255] For example, incubation for initial expansion of T cells in the input sample can be performed under GMP conditions. In some embodiments, the incubation is in a closed system, which in some aspects can be a closed, automated system. In some embodiments, the culture medium containing the T cell stimulator can be serum-free medium. In some embodiments, the incubation is performed using serum-free medium in a closed, automated system.

[0256] In some embodiments, the initial expansion of cells under one or more stimulus 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 100M / 100M-CS, or G-Rex 500M / 500M-CS). In some embodiments, the culture vessel is a microplate, flask, bar, or other culture vessel suitable for expanding cells in a closed system. Bioreactor: In some embodiments, expansion may occur in a bioreactor. In some embodiments, initial expansion may occur using a cell expansion system, for example, by transferring cells to a gas-permeable bag in connection with a bioreactor (e.g., Xuri Cell Expansion System W25 (GE Healthcare)). In one embodiment, the cell expansion system includes a culture vessel, such as a bag, e.g., a gas-permeable cell bag, having a volume of 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 bioreactors suitable for automated perfusion expansion include, but are not limited to, the 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 performed under static conditions. In some embodiments, the expansion culture is performed under rocking conditions. Media can be added in a bolus or on a perfusion schedule.In some embodiments, the bioreactor has a flow rate of 0.01 L / min, about 0.01 L / min, or at least 0.01 L / min, 0.05 L / min, about 0.05 L / min, or at least 0.05 L / min, 0.1 L / min, about 0.1 L / min, or at least 0.1 L / min, 0.2 L / min, about 0.2 L / min, or at least 0.2 L / min, 0.3 L / min, about 0.3 L / min, or at least 0.3 L / min, 0.4 L / min, about 0.4 L / min, or at least The temperature is maintained at or near 37°C and the CO2 level is maintained at or near 5% using a constant airflow of at least 0.4 L / min, 0.5 L / min, about 0.5 L / min, or at least 0.5 L / min, 1.0 L / min, about 1.0 L / min, or at least 1.0 L / min, 1.5 L / min, about 1.5 L / min, or at least 1.5 L / min, or 2.0 L / min, about 2.0 L / min, or at least 2.0 L / min, or greater than 2.0 L / min. In certain embodiments, at least a portion of the culture is performed using perfusion, e.g., using a rate of 290 ml / day, 580 ml / day, and / or 1160 ml / day.

[0257] In some embodiments, the cells are 0.5 x 10 cells 6 cells / mL~cells 1.5×10 6 The cells are seeded into a suitable culture vessel (e.g., a gas-permeable bag) at a density of 0.5 x 10 cells / mL. In some embodiments, the density is 0.5 x 10 cells / mL. 6 cells / mL, cells 0.75×10 6 cells / mL, cells 1×10 6 cells / mL, cells 1.25×10 6 cells / mL or 1.5 x 10 cells 6 cells / mL, or approximately 0.5 x 10 cells 6 cells / mL, cells approximately 0.75×10 6 cells / mL, approximately 1×10 cells 6 cells / mL, approximately 1.25×10 cells 6 cells / mL or approximately 1.5 x 10 cells 6 cells / mL, or any value between any of the aforementioned.

[0258] In some aspects, cells are expanded in an automated, closed expansion system capable of perfusion. The perfusion fluid can continuously add medium to the cells to ensure optimal growth rates are achieved.

[0259] The expansion method can be performed in a closed, automated system and under GMP conditions, including using serum-free media. In some embodiments, any one or more of the method steps can be performed in a closed system or under GMP conditions. In certain embodiments, the entire process is performed in a GMP suite. In some embodiments, a closed system is used to perform one or more of the other processing steps of the method for manufacturing, producing, or creating a cell therapy. In some embodiments, one or more or all of the processing steps, such as isolation, selection and / or enrichment, processing, culturing steps including incubation associated with expanding cells, and formulation steps, are performed in an integrated or self-contained system and / or using a system, device, or instrument in an automated or programmable manner. In some aspects, the system or instrument includes a computer and / or computer program in communication with the system or instrument, allowing a user to program, control, evaluate the results of, and / or adjust various aspects of the processing, isolation, manipulation, and formulation steps.

[0260] In some embodiments, immediately after incubation, stimulated cells can be collected for subsequent co-culture with APCs, for example, according to the methods described in Section IB2 below.

[0261] In some embodiments, stimulated cells are collected and cryofrozen. Cryopreservation, providing an intermediate storage step after the initial expansion stage, can be used to time neoepitope identification and peptide generation, as described in Section IB1, and / or APC generation, as described in Section IB2. In some embodiments, for cryopreservation, stimulated cells are formulated as a composition comprising a cryoprotectant. In some embodiments, the cryoprotectant is or comprises DMSO and / or glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, e.g., ultra-low temperatures, e.g., between -40°C and -150°C, e.g., at or about 80°C ± 6.0°C.

[0262] In some embodiments, cryopreserved cells are prepared for subsequent processing by thawing. In some cases, cells may be ready for subsequent culture with APCs and peptides immediately after thawing after one or more washing steps.

[0263] B. Neoantigen Identification and Co-culture of T Cells with APCs In certain embodiments of the provided methods, the methods may include co-culturing a tumor-reactive T cell population with antigen-presenting cells that present one or more neo-antigenic peptides corresponding to associated non-synonymous somatic mutations in the subject's tumor. In other aspects of the provided methods, the co-culturing step further enriches tumor-reactive T cells, since T cells that recognize neo-antigenic peptides derived from sequencing data obtained from the patient's tumor can be activated. Methods for co-culturing may include methods for identifying neo-epitopes and generating peptides, followed by contacting the peptides with antigen-presenting cells for presentation to a T cell population known to contain or suspected to contain tumor-reactive T cells.

[0264] In some embodiments, after the co-culture step, selection can be performed for upregulation of activation markers. In some cases, selection can be for cells positive for any upregulation marker as described herein (e.g., PD-1, CD39, and / or TIGIT or other upregulation markers). Cells from the culture are then expanded to create a therapeutic composition containing an expanded tumor-specific reactive cell population.

[0265] In other cases, the selection of cells after co-culture can be the second selection in the process of enriching and expanding tumor-reactive T cells. For example, in some embodiments, cells directly digested from tumor fragments from a subject, or cells after the initial (e.g., minimal expansion) expansion of cells from tumor fragments from a subject, are enriched or selected (e.g., based on the selection of cells positive for PD-1, CD39, and / or TIGIT), and the selected T cell population is co-cultured with antigen-presenting cells that present peptide epitopes. After co-culture incubation, the cells from the co-culture can be further enriched for tumor-reactive T cells, for example, by selecting any up-regulated marker described herein or a combination thereof, such as 4-1BB (CD137) or OX40 (CD134) or 4-1BB (CD137) and OX40 (CD134) double-positive cells. The cells from the culture are then expanded to create a therapeutic composition containing an expanded tumor-specific reactive cell population.

[0266] 1. Neoepitope identification and peptide generation In some ...

Claims

1. a. selecting cells from an input sample comprising T cells from a subject with a tumor that are surface positive for at least two activation markers selected from the group consisting of PD-1, CD39, and TIGIT to obtain selected cells from the sample; and b. Expanding by culturing the selected cells with one or more T cell stimulants of lymphocytes under conditions to produce an expanded T cell population. A method for producing tumor-reactive T cells, comprising:

2. The method of claim 1, wherein the selecting step is for cells that are surface positive for PD-1 and CD39, PD-1 and TIGIT, or CD39 and TIGIT.

3. The method described in claim 1 or 2, wherein the selecting step is for cells that are surface positive for PD-1, CD39, and TIGIT.

4. 4. The method of claim 1, wherein the selecting step further comprises selecting cells surface-positive for one or more additional markers selected from CD107a, CD39, CD103, CD137 (4-1BB), CD59, CD90, CD36, CD38, CD30, CD154 (CD40L), CD134 (OX40), CD152 (CTLA-4), CD160, CXCR5 (CD195), CD244, CD258 (LIGHT), CD256 (APRIL), CD272 (BTLA-4), PD-1, TIM-3, LAG-3, or TIGIT, and wherein the step of selecting cells surface-positive for the activation markers and the step of selecting cells surface-positive for the additional markers are performed simultaneously or sequentially in any order to obtain the selected cells.

5. (i) the input sample contains tumor-infiltrating lymphocytes, and / or (ii) the input sample containing T cells is derived from peripheral blood or from a tumor; 5. The method of any one of claims 1 to 4.

6. 6. The method of any one of claims 1 to 5, wherein the input sample containing T cells is derived from a resected tumor.

7. 7. The method of claim 6, wherein the input sample containing T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor.

8. The method of any one of claims 1 to 7, wherein the expanded T cell population is for use as a therapeutic cell composition.

9. performing said expanding to generate said expanded T cell population, (i) for 7 to 35 days; (ii) lasting between 7 and 28 days; (iii) for 14 to 28 days; (iv) for 7 to 21 days; and / or (v) for 7 to 14 days; 9. The method of any one of claims 1 to 8.

10. 10. The method of any one of claims 1 to 9, wherein the one or more T cell stimulators are one or more first T cell stimulators, the expanding step is a first expansion, and the method further comprises a second expansion step by culturing the first expanded T cell population with one or more second T cell stimulators under conditions to generate a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

11. 11. The method of claim 10, wherein said first expansion step and / or said second expansion step is for a period of 7 to 21 days, optionally 7 to 14 days.

12. 12. The method of any one of claims 1 to 11, wherein the one or more T cell stimulants of lymphocytes are an anti-CD3 substance (e.g. OKT3) and / or a recombinant cytokine selected from one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and IL-35.

13. 13. The method of any one of claims 1 to 12, wherein at least one of said one or more T cell stimulators is recombinant IL-2.

14. a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. selecting cells from the first expanded cell population that are surface positive for two or more activation markers to generate a selected population; and d. performing a second expansion by culturing the selected population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition.

2. The method of claim 1, comprising:

15. a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. selecting cells from the first expanded cell population that are surface positive for at least two activation markers from the group consisting of PD-1, CD39, and TIGIT to generate a selected population; and d. performing a second expansion by culturing the selected population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. A method for producing tumor-reactive T cells, comprising:

16. a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. selecting cells from the first expanded cell population that are surface positive for at least two activation markers from the group consisting of PD-1, CD39, and TIGIT to generate a selected cell population; and d. co-culturing the selected cell population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells that comprise endogenous T cell receptors that are reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-native peptides are peptides that correspond to an associated non-synonymous somatic mutation in the tumor of interest; e. performing a second expansion by culturing the reactive T cell population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. A method for producing tumor-reactive T cells, comprising:

17. a. processing a biological sample containing T cells obtained from a tumor-bearing donor subject to generate an input sample containing T cells; b. performing a first expansion by culturing said sample containing T cells with one or more first T cell stimulants of lymphocytes under conditions to produce a first expanded T cell population; c. co-culturing the first expanded cell population in the presence of antigen-presenting cells that present one or more non-native peptides on the major histocompatibility complex (MHC) to generate a reactive T cell population comprising T cells comprising an endogenous T cell receptor that is reactive to a peptide-encoding mutation of the tumor, wherein the one or more non-native peptides are peptides that correspond to an associated non-synonymous somatic mutation in the tumor of interest; d. selecting cells from the reactive T cell population that are surface positive for at least two activation markers from the group consisting of PD-1, CD39, and TIGIT to generate a selected cell population; and e. performing a second expansion by culturing the selected cell population with one or more second T cell stimulators under conditions to produce a second expanded T cell population, wherein the second expanded T cell population is for use as a therapeutic cell composition. A method for producing tumor-reactive T cells, comprising:

18. A method described in any one of claims 15 to 17, wherein the selecting step is for cells that are surface positive for PD-1 and CD39, PD-1 and TIGIT, or CD39 and TIGIT.

19. A method described in any one of claims 15 to 17, wherein the selecting step is for cells that are surface positive for PD-1, CD39, and TIGIT.

20. 20. The method of any one of claims 1 to 19, further comprising the step of selecting, optionally by positive or negative selection, surface-positive T cells for a T cell marker selected from CD3, CD4, or CD8, wherein the steps of selecting cells surface-positive for said T cell marker and selecting cells surface-positive for said activation marker are performed simultaneously or sequentially in any order to obtain said selected cells and / or to generate said selected cell population.

21. 21. The method of any one of claims 1 to 20, wherein the input sample comprising T cells is derived from a resected tumor.

22. the biological sample is a resected tumor from the subject; (i) the input sample containing T cells is one or more tumor fragments from the resected tumor; or (ii) the input sample containing T cells is a single cell suspension processed by homogenization and / or enzymatic digestion of one or more tumor fragments from the resected tumor; 22. The method of any one of claims 1 to 21.

23. The concentration of recombinant IL-2 is (i) 100 IU / mL to 6000 IU / mL; (ii) 300 IU / mL to 1000 IU / mL, optionally with a recombinant IL-2 concentration of 300 IU / mL or about 300 IU / mL; (iii) at or about 300 IU / mL; and / or (iv) 1000 IU / mL or about 1000 IU / mL; 14. The method of claim 12 or 13.

24. A method described in any one of claims 1 to 23, wherein the step of selecting cells surface-positive for the activation marker comprises contacting cells from an input sample containing T cells with an antibody that binds to the activation marker to obtain selected cells from the sample, and selecting cells surface-positive for the activation marker.

25. 25. The method of any one of claims 1 to 24, wherein the step of selecting the cells is carried out using a fluorescence-based cell sorter.

26. 14. The method of any one of claims 1 to 13, wherein said expanding step is carried out in a closed system using a gas permeable membrane, using a bioreactor, or using a gas permeable culture vessel.

27. the tumor is (i) The tumor is an epithelial cancer; (ii) the tumor is melanoma, squamous cell lung carcinoma, adenocarcinoma of the lung, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer (CRC), cervical cancer, head and neck cancer, gastric cancer, or uterine cancer; (iii) melanoma; (iv) colorectal cancer (CRC); and / or (v) The tumor is non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer; 27. The method of any one of claims 1 to 26.

28. 28. The method of any one of claims 1 to 27, further comprising harvesting the cells produced by said method for formulation as a therapeutic composition.

29. 29. A composition comprising tumor-reactive T cells generated by the method of any one of claims 1 to 28.

30. 30. The composition of claim 29, comprising a cryoprotectant.

31. 31. The composition of claim 29 or 30 for treating a subject with cancer.

32. 32. The composition of claim 31, wherein the cells of the administered composition are autologous to the subject.

33. The therapeutically effective dose is (i) 1 × 10 8 ~10×10 9 individual T cells or live T cells, (ii) 5×10 8 to 10×10 9 T cells or live T cells thereof; and / or (iii) 5×10 8 to 1×10 9 T cells or live cells thereof; 33. The composition of claim 31 or 32.

34. The cancer is (i) It is an epithelial cancer; (ii) melanoma, squamous cell lung carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer; and / or (iii) non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer; 34. The composition of any one of claims 31 to 33.

35. 35. Use of the composition of any one of claims 29 to 34 for the manufacture of a medicament for treating a subject with cancer.

36. 36. The use of claim 35, wherein the cells of the administered composition are autologous to the subject.

37. The therapeutically effective dose is (i) 1 × 10 8 ~10×10 9 individual T cells or live T cells, (ii) 5×10 8 to 10×10 9 T cells or live T cells thereof; and / or (iii) 5×10 8 to 1×10 9 T cells or live cells thereof; 37. Use according to claim 35 or 36.

38. The cancer is (i) It is an epithelial cancer; (ii) melanoma, squamous cell lung carcinoma, lung adenocarcinoma, bladder cancer, small cell lung carcinoma, esophageal cancer, colorectal cancer, cervical cancer, head and neck cancer, gastric cancer, or uterine cancer; and / or (iii) non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, or endometrial cancer, and optionally, the breast cancer is HR+ / Her2- breast cancer, triple-negative breast cancer (TNBC), or HER2+ breast cancer; 38. Use according to any one of claims 35 to 37.

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