Compositions and methods for generating neoantigen-reactive tumor-infiltrating lymphocytes

Abstract

Various aspects of the present invention provide compositions of tumor-infiltrating lymphocytes (TILs) enriched for tumor-reactive cells, methods for producing TILs enriched for tumor-reactive cells, and methods and uses of the provided enriched tumor-reactive TILs for treating cancer in humans or other subjects. Among the provided aspects of the TIL compositions can be TIL compositions that exhibit substantial tumor-reactive activity, including degranulation and the ability to express one or more of IFN-γ and TNF-α in response to antigen-presenting cells that present neo-antigenic peptides. TIFF2025537148000002.tif51170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 422,914, filed November 4, 2022, entitled "COMPOSITIONS AND METHODS FOR GENERATING NEO-ANTIGEN REACTIVE TUMOR INFILTRATING LYMPHOCYTES," U.S. Provisional Patent Application No. 63 / 490,000, filed March 14, 2023, entitled "COMPOSITIONS AND METHODS FOR GENERATING NEO-ANTIGEN REACTIVE TUMOR INFILTRATING LYMPHOCYTES," and U.S. Provisional Patent Application No. 63 / 594,405, filed October 30, 2023, entitled "COMPOSITIONS AND METHODS FOR GENERATING NEO-ANTIGEN REACTIVE TUMOR INFILTRATING LYMPHOCYTES," the contents of which are incorporated by reference in their entireties.

[0002] Field Embodiments of the present invention relate to compositions of tumor-infiltrating lymphocytes (TILs) enriched for tumor-reactive cells. Embodiments of the present invention also relate to methods for producing TILs enriched for tumor-reactive cells, and to the use of the provided enriched tumor-reactive TILs for treating cancer in a subject. [Background technology]

[0003] background The process of making autologous tumor-infiltrating lymphocyte (TIL) therapy involves a small number of reactive cells, resulting in a composition that is not completely suitable for therapeutic use, including the treatment of cancer.Therefore, it is considered desirable to develop an improvement to TIL composition to overcome these limitations.The present specification provides an embodiment that meets this need. Summary of the Invention

[0004] Various aspects of the present invention provide pharmaceutical T lymphocyte infiltrating (TIL) compositions enriched in tumor-reactive T cells, methods for producing TIL compositions enriched in tumor-reactive T cells, and methods of using such compositions for various therapies, including for the treatment of cancer and other diseases.

[0005] According to one embodiment, a pharmaceutical composition comprises a multiclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein the population is enriched for at least 10 different T cell receptor (TCR) clonotypes, each having a frequency of at least 1.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells. According to one embodiment, a pharmaceutical composition comprises an oligoclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein the population is enriched for at least 10 different T cell receptor (TCR) clonotypes, each having a frequency of at least 1.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells. According to one embodiment, a pharmaceutical composition comprises a multiclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein the population is enriched for at least 10 different T cell receptor (TCR) clonotypes, each having a frequency of at least 2.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells. According to one embodiment, a pharmaceutical composition comprises an oligoclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, the population enriched for at least 10 different T cell receptor (TCR) clonotypes, each having a frequency of at least 2.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells. Various embodiments of the invention are particularly useful in treating difficult-to-treat cancers, including cancers that have mutations unique to a particular patient, including instances where such mutations render the cancer resistant or refractory to standard cancer treatments (e.g., chemotherapy).

[0006] According to one or more embodiments, a multiclonal population of T cells has a minimum number of different TCR clonotypes that constitute a selected percentage of the population. According to one or more embodiments, an oligoclonal population of T cells has a minimum number of different TCR clonotypes that constitute a selected percentage of the population. For example, in one or more embodiments, at least 11 different TCR clonotypes have a frequency of at least 2.0% in the population, or at least 12 different TCR clonotypes have a frequency of at least about 2.0% in the population. Further, in one or more embodiments, at least 11 different TCR clonotypes have a frequency of at least 1.0% in the population, or at least 12 different TCR clonotypes have a frequency of at least about 1.0% in the population. According to some embodiments, 8 to 15 different T cell receptor (TCR) clonotypes constitute at least about 50% of the TCR frequencies in the population.

[0007] According to some embodiments, pharmaceutical compositions enriched for tumor-reactive T cells comprise a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 8-15 different T cell receptor (TCR) clonotypes comprise at least 50% of the TCR frequency within the population, with increasing or decreasing percentages of contemplated clonotypes and populations. According to some embodiments, pharmaceutical compositions enriched for tumor-reactive T cells comprise an oligoclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 8-15 different T cell receptor (TCR) clonotypes comprise at least 50% of the TCR frequency within the population, with increasing or decreasing percentages of contemplated clonotypes and populations. According to some embodiments, 9-12 different TCR clonotypes comprise at least 50% of the TCR frequency within the population.

[0008] In various embodiments, the TCR clonotypes exhibit reactivity to at least one CD4 antigen and at least one CD8 antigen. In some of the provided embodiments, the TCR clonotypes exhibit reactivity to 2-100 different peptide antigens. In some of the provided embodiments, the TCR clonotypes exhibit reactivity to 10-40 different peptide antigens. In some of the provided embodiments, the TCR clonotypes exhibit reactivity to 2-6 different peptide antigens. In some of the provided embodiments, the TCR clonotypes exhibit reactivity to 2-4 different peptide antigens. In some of the provided embodiments, the TCR clonotypes exhibit reactivity to two different peptide antigens. In some of the provided embodiments, the TCR clonotypes exhibit reactivity to one CD8 antigen and one CD4 antigen.

[0009] Provided herein are pharmaceutical compositions enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 10 to 100 different T cell receptor (TCR) clonotypes are present within the population. In some of any of the provided embodiments, the TCR clonotypes exhibit reactivity to 10 to 40 different peptide antigens.

[0010] Provided herein are pharmaceutical compositions enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 20 to 100 different T cell receptor (TCR) clonotypes are present within the population. In some of any of the provided embodiments, 20 to 60 different TCR clonotypes are present within the population.

[0011] Provided herein is a pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein the top 40 TCR clonotypes comprise at least 75% of the TCR frequency in the population. In some of the provided embodiments, at least 90% of the cells in the population are CD3+ T cells. In some of the provided embodiments, the TCR clonotypes exhibit reactivity to at least one CD8 antigen and at least one CD4 antigen.

[0012] In some of any of the provided embodiments, at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

[0013] Various embodiments provide pharmaceutical T-lymphocyte infiltration (TIL) compositions enriched for tumor-reactive T cells, comprising tumor-infiltrating lymphocytes, including tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity. In some of any of the provided embodiments, at least 40% of the CD8+ T cells and / or at least 30% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

[0014] In one or more embodiments, at least 25% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity. In some of the provided embodiments, at least 30% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity. In some of the provided embodiments, neoantigen reactivity is determined after co-culture with peptide-loaded antigen-presenting cells and is characterized by one or more of IFN-γ production or degranulation. In some embodiments, degranulation is determined based on CD107 expression.

[0015] In some of the provided embodiments, neoantigen reactivity is determined in a co-culture assay with autologous APCs and neoantigen peptides (e.g., as described in Example 2) by one or more of upregulation of CD134 and CD137, IFN-γ production, TNF-α production, granzyme B production, or degranulation. In some embodiments, degranulation is determined based on CD107 expression. In some of the provided embodiments, the TIL composition is characterized by at least a 1.5-fold increase in the percentage of cells positive for CD134 and CD137 compared to the bulk TIL population in a co-culture assay with autologous APCs and neoantigen peptides. In some embodiments, the TIL composition is characterized by at least a 2-fold, at least a 3-fold, or at least a 4-fold increase in CD134-positive cells and CD137-positive cells compared to the bulk TIL population. In some of any of the provided embodiments, in a co-culture assay with peptide-loaded autologous APCs, more than 30% of the cells in the TIL composition are positive for CD134 and CD137, and optionally, more than about 35%, more than about 40%, or more than about 45% of the cells are positive for CD134 and CD137.

[0016] In some of any of the provided embodiments, greater than 48% of the cells in the TIL composition are positive for CD 134 and CD 137 in a co-culture assay with autologous APCs and the neo-antigenic peptide. In some embodiments, greater than about 50%, greater than about 60%, or greater than about 70% of the cells are positive for CD 134 and CD 137 in a co-culture assay with autologous APCs and the neo-antigenic peptide.

[0017] In some of any of the provided embodiments, the TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ production greater than 1,000 pg / mL; (ii) TNF-α production greater than 100 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) granzyme B production greater than 10,000 pg / mL.

[0018] In some of any of the provided embodiments, the TIL composition is characterized in an in vitro co-culture assay with autologous APCs and neoantigenic peptides by at least one of the following: (i) IFN-γ production greater than 100,000 pg / mL; (ii) TNF-α production greater than 250 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) granzyme B production greater than 50,000 pg / mL.

[0019] Provided herein is a pharmaceutical T-lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, wherein the pharmaceutical composition comprises tumor-infiltrating lymphocytes, including tumor-derived CD4+ T cells and CD8+ T cells, and at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized in an in vitro coculture assay with peptide-loaded autologous APCs by at least one of the following criteria: (i) IFN-γ production greater than 1,000 pg / mL; (ii) TNF-α production greater than 100 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) granzyme B production greater than 10,000 pg / mL.

[0020] Provided herein is a pharmaceutical T-lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, wherein the pharmaceutical composition comprises tumor-infiltrating lymphocytes, including tumor-derived CD4+ T cells and CD8+ T cells, and at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized in an in vitro co-culture assay by at least one of the following: (i) IFN-γ production greater than 100,000 pg / mL; (ii) TNF-α production greater than 250 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) granzyme B production greater than 50,000 pg / mL.

[0021] In some of the provided embodiments, the TIL composition is characterized by at least two of (i)-(iv). In some of the provided embodiments, the TIL composition is characterized by at least three of (i)-(iv). In some of the provided embodiments, the TIL composition is characterized by (i)-(iv).

[0022] In some of any of the provided embodiments, the TIL compositions are characterized by IFN-γ production of greater than 2,500 pg / mL, greater than 5,000 pg / mL, greater than 10,000 pg / mL, greater than 25,000 pg / mL, greater than 50,000 pg / mL, greater than 100,000 pg / mL, greater than 200,000 pg / mL, greater than 250,000 pg / mL, greater than 500,000 pg / mL, or greater than 1,000,000 pg / mL. In some of any of the provided embodiments, the TIL compositions are characterized by IFN-γ production of greater than 250,000 pg / mL, greater than 500,000 pg / mL, or greater than 1,000,000 pg / mL. In some of the provided embodiments, the TIL compositions are characterized by IFN-γ production of greater than 250,000 pg / mL, greater than 500,000 pg / mL, or greater than 1,000,000 pg / mL. In some of the provided embodiments, the TIL compositions are characterized by TNF-α production of greater than 200 pg / mL, greater than 500 pg / mL, greater than 1000 pg / mL, or greater than 2000 pg / mL. In some of the provided embodiments, the TIL compositions are characterized by TNF-α production of greater than 500 pg / mL, greater than 1000 pg / mL, or greater than 2000 pg / mL.

[0023] In some of the provided embodiments, the TIL compositions are characterized by greater than 15% CD107a+ cells, greater than 20% CD107a+ cells, or greater than 25% CD107a+ cells. In some of the provided embodiments, the TIL compositions are characterized by Granzyme B production of greater than 15,000 pg / mL, greater than 25,000 pg / mL, greater than 50,000 pg / mL, greater than 100,000 pg / mL, greater than 200,000 pg / mL, greater than 300,000 pg / mL, greater than 400,000 pg / mL, or greater than 500,000 pg / mL. In some of the provided embodiments, the TIL compositions are characterized by Granzyme B production of greater than 200,000 pg / mL, greater than 300,000 pg / mL, greater than 400,000 pg / mL, or greater than 500,000 pg / mL.

[0024] In some of any of the provided embodiments, after co-culture with peptide-loaded antigen-presenting cells, the TIL composition is characterized by at least one of the following: (i) IFN-γ secretion in the supernatant that is 50 times or more higher than that of a bulk TIL composition not enriched for tumor-reactive T cells; (ii) TNF-α secretion in the supernatant that is 300 times or more higher than that of a bulk TIL composition not enriched for tumor-reactive T cells; or (iii) granzyme B secretion in the supernatant that is 15 times or more higher than that of a bulk TIL composition not enriched for tumor-reactive T cells.

[0025] In some of any of the provided embodiments, the TIL composition is characterized by at least one of the following criteria as determined in and / or by an in vitro coculture assay: (i) IFN-γ that is 50-fold or more than that of a bulk TIL composition not enriched for tumor-reactive T cells; (ii) TNF-α that is 300-fold or more than that of a bulk TIL composition not enriched for tumor-reactive T cells; or (iii) granzyme B that is 15-fold or more than that of a bulk TIL composition not enriched for tumor-reactive T cells.

[0026] In many embodiments, a pharmaceutical T lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells comprises tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and after co-culture with peptide-loaded antigen-presenting cells, the TIL composition is characterized by at least one of the following criteria: (i) IFN-γ secretion in the supernatant that is 50-fold or more higher than that of a bulk TIL composition not enriched for tumor-reactive T cells; (ii) TNF-α secretion in the supernatant that is 300-fold or more higher than that of a bulk TIL composition not enriched for tumor-reactive T cells; or (iii) granzyme B secretion in the supernatant that is 15-fold or more higher than that of a bulk TIL composition not enriched for tumor-reactive T cells.

[0027] In many embodiments, a pharmaceutical T lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells comprises tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized by at least one of the following criteria as determined in and / or by an in vitro co-culture assay: (i) IFN-γ that is 50-fold or more than that of a bulk TIL composition not enriched for tumor-reactive T cells; (ii) TNF-α that is 300-fold or more than that of a bulk TIL composition not enriched for tumor-reactive T cells; or (iii) granzyme B that is 15-fold or more than that of a bulk TIL composition not enriched for tumor-reactive T cells.

[0028] In some of the provided embodiments, the TIL composition is characterized by criteria (i) and (ii). In some of the provided embodiments, the TIL composition is characterized by criteria (i) and (iii). In some of the provided embodiments, the TIL composition is characterized by criteria (ii) and (iii). In some of the provided embodiments, the TIL composition is characterized by criteria (i), (ii), and (iii).

[0029] In some of the provided embodiments, the composition features a greater number of CD4+ T cells than CD8+ T cells. In some of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 1:5. In some of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:1. In some of any of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is between 10:1 and 25:1, or about 10:1 and 25:1. In some embodiments, the ratio of CD4+ T cells to CD8+ T cells is 20:1 or about 20:1.

[0030] In some of any of the provided embodiments, greater than 50% of the CD3+ T cells, and optionally greater than 50% of the CD4+ T cells and CD8+ T cells, express markers for an effector memory phenotype. According to various embodiments, greater than 75% of the CD3+ T cells, and optionally greater than 75% of the CD4+ T cells and CD8+ T cells, express markers for an effector memory phenotype. In some of the provided embodiments, greater than 80% of the CD3+ T cells, and optionally greater than 80% of the CD4+ T cells and CD8+ T cells, express markers for an effector memory phenotype. In some of the provided embodiments, greater than 85% of the CD3+ T cells, and optionally greater than 85% of the CD4+ T cells and CD8+ T cells, express markers for an effector memory phenotype. In some of the provided embodiments, greater than 90% of the CD3+ T cells, and optionally greater than 90% of the CD4+ T cells and CD8+ T cells, express markers for an effector memory phenotype. In some of any of the provided embodiments, the effector memory phenotype is characterized by surface marker expression that is one or more of CD45RO+, CD45RA-, CD62L-, CCR7-, CD28-, and CD27-. In some of any of the provided embodiments, the effector memory phenotype is characterized by surface marker expression CD45RO+, CD45RA-, CD62L-, and CCR7-. In some of any of the provided embodiments, the effector memory phenotype is characterized by surface marker expression CD45RO+, CD45RA-, CD62L-, CCR7-, CD28-, and CD27-. In some of any of the provided embodiments, the effector memory phenotype is characterized by surface marker expression CD45RA - and CCR7 -- In some of the provided embodiments, greater than 95% of the CD4+ T cells and CD8+ T cells in the composition are PD-1-. In some of the provided embodiments, greater than 80% of the CD4+ T cells and CD8+ T cells in the composition are LAG3-.

[0031] In some of any of the provided embodiments, the number of cells in the composition, or the number of viable cells thereof, is at least 2 x 10 7In some of any of the provided embodiments, the number of cells in the composition, or the number of viable cells thereof, is 2 x 10 7 pieces~20×10 9 pieces, 2×10 7 pieces~10×10 9 pieces, 2×10 7 pieces~2×10 9 pieces, 2×10 7 pieces~2×10 8 pieces, 2×10 8 pieces~20×10 9 pieces, 2×10 8 pieces~10×10 9 pieces, 2×10 8 pieces~2×10 9 pieces, 2×10 9 pieces~20×10 9 pieces, 2×10 9 pieces~10×10 9 pieces, or 10 x 10 9 pieces~20×10 9 pieces, or approximately 2 x 10 7 pieces~20×10 9 pieces, approximately 2×10 7 pieces~10×10 9 pieces, approximately 2×10 7 pieces~2×10 9 pieces, approximately 2×10 7 pieces~2×10 8 pieces, approximately 2×10 8 pieces~20×10 9 pieces, approximately 2×10 8 pieces~10×10 9 pieces, approximately 2×10 8 pieces~2×10 9 pieces, approximately 2×10 9 pieces~20×10 9 pieces, approximately 2×10 9 pieces~10×10 9 pieces, or about 10 x 10 9 pieces~20×10 9 (inclusive).

[0032] In some of the provided embodiments, the pharmaceutical composition is for treating a tumor in a patient. In some of the provided embodiments, the tumor is a colorectal cancer (CRC) tumor. In some of the provided embodiments, the tumor is a colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor. In some of the provided embodiments, the tumor is derived from a human subject. In some of the provided embodiments, the pharmaceutical composition is for autologous adoptive therapy for a human subject. In some of the provided compositions, a pharmaceutically acceptable excipient is included. Provided herein are any of the provided compositions that include a cryoprotectant.

[0033] In some of the provided embodiments, the composition is a liquid composition. In some of the provided embodiments, the composition has been frozen and thawed. In some of the provided embodiments, the volume of the composition is between 1 mL and 500 mL. In some of the provided embodiments, the composition is frozen.

[0034] Many embodiments provide a pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched in tumor-reactive T cells for treating a tumor in a patient, the pharmaceutical composition comprising a multiclonal population of T cells comprising CD4+ T cells and CD8+ T cells from the patient's tumor, the population enriched for at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 1.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells. Further embodiments provide a pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched in tumor-reactive T cells for treating a tumor in a patient, the pharmaceutical composition comprising a multiclonal population of T cells comprising CD4+ T cells and CD8+ T cells from the patient's tumor, the population enriched for at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 2.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells.

[0010] Another embodiment provides a pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched in tumor-reactive T cells for treating a tumor in a patient, the pharmaceutical composition comprising an oligoclonal population of T cells comprising CD4+ T cells and CD8+ T cells from the patient's tumor, the population being enriched for at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 2.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells. Also provided herein is a pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched in tumor-reactive T cells for treating a tumor in a patient, the pharmaceutical composition comprising an oligoclonal population of T cells comprising CD4+ T cells and CD8+ T cells from the patient's tumor, the population being enriched for at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 1.0% in the population, and at least 90% of the cells in the composition are CD3+ T cells.

[0035] In some of any of the provided embodiments, the TIL composition is generated by an ex vivo method involving the expansion of tumor-reactive T cells from a donor subject, which are co-cultured with autologous antigen-presenting cells and peptide neoantigens.

[0036] In some of the provided embodiments, the TIL composition comprises: a. providing tumor cells dissociated from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first T cell population comprising CD4+ T cells and CD8+ T cells; b. culturing the first T cell population with recombinant IL-2 added at a concentration of 3000 IU / mL to 6000 IU / mL (inclusive) for 14 to 28 days to generate a second T cell population; c. co-culturing the second T cell population with autologous antigen-presenting cells (APCs) for 12 to 48 hours with recombinant IL-2 added at a concentration of 100 IU / mL to 1000 IU / mL to generate a third T cell population, wherein the APCs are loaded with a pool of tumor-derived peptide neoantigens (i.e., neoantigen peptides), each peptide being 13 to 40 amino acids in length and loaded at a concentration of 100 ng / mL per peptide, at a ratio of the second T cell population to the APCs of 2:1 to 10:1; d. selecting cells that are surface-positive for CD134 and / or CD137 from the third T cell population to generate a fourth T cell population; and e. To produce a therapeutic composition of TILs enriched for tumor-reactive cells, the composition is produced by a method comprising the step of expanding tumor-infiltrating lymphocytes (TILs) by incubating irradiated human peripheral blood mononuclear cells (iPBMCs) and a fourth T cell population at a ratio of 100:500 iPBMCs to cells of the fourth T cell population for 12 to 16 days with recombinant IL-2 added at a concentration of 3000 IU / mL to 6000 IU / mL (inclusive) and 10 to 50 ng / mL of anti-CD3 antibody (OKT3).

[0037] Provided herein is a cryocomposition comprising any of the pharmaceutical compositions provided and a cryoprotectant.

[0038] Provided herein is a method for producing a T lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells, the method comprising the following steps: a. providing tumor cells dissociated from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first T cell population comprising CD4+ T cells and CD8+ T cells; b. culturing the first T cell population with recombinant IL-2 added at a concentration of about 3000 IU / mL to 6000 IU / mL (inclusive) for about 14 to 28 days to produce a second T cell population; c. co-culturing the second T cell population with autologous antigen-presenting cells (APCs) for about 12 to 48 hours with recombinant IL-2 added at a concentration of 100 IU / mL to 1000 IU / mL to generate a third T cell population, wherein the APCs are loaded with a pool of tumor-derived peptide neoantigens (i.e., neoantigenic peptides), each peptide being 13 to 40 amino acids in length and loaded at a concentration of 100 ng / mL per peptide, at a ratio of the second T cell population to the APCs of about 2:1 to 10:1; d. selecting cells from the third T cell population that are surface-positive for CD134 and / or CD137 to generate a fourth T cell population; and e. To generate a therapeutic composition of TILs enriched for tumor-reactive cells, the step of expanding tumor-infiltrating lymphocytes (TILs) by incubating irradiated human peripheral blood mononuclear cells (iPBMCs) with the fourth T cell population at a ratio of about 100:500 iPBMCs to cells of the fourth T cell population for 12 to 16 days with recombinant IL-2 added at a concentration of about 3000 IU / mL to 6000 IU / mL (inclusive) and 10 to 50 ng / mL of anti-CD3 antibody (OKT3).

[0039] Provided herein are methods for treating a subject with cancer, comprising administering to the subject having a tumor a therapeutic dose of any of the provided compositions. In some of the provided embodiments, the therapeutically effective dose is about 1×10 9 ~10×10 9In some of the embodiments provided, the therapeutically effective dose is from greater than 1 million to less than 100 million T cells per kilogram of body weight. In some of the embodiments provided, the therapeutically effective dose is from greater than 1 million to less than 10 million T cells per kilogram of body weight. In some of the embodiments provided, the therapeutically effective dose is from 10 million or about 10 million to 50 million or about 50 million T cells per kilogram of body weight. In some of the embodiments provided, the cells of the therapeutic composition are autologous to the subject. [Brief explanation of the drawings]

[0040] [Figure 1] We demonstrate aspects of a process for preparing enriched tumor-reactive TILs, including unbiased mutation calling using peptides generated against a broad range (up to 200) of relevant tumor neoantigens, co-culture of TILs prepared from dissociated tumor cells of CRC patients with autologous antigen-presenting cells to optimize peptide-based antigen presentation and capture maximum TCR diversity within the TIL population, and selective sorting of cells with validated markers such as CD134 and / or CD137 to enrich for both CD4+ and CD8+ TILs with the highest tumor reactivity. [Figure 2] Figure 2A shows tumor mutation burden (TMB) across multiple indications. The number of tumor-specific neoantigens (i.e., neoantigenic peptides) identified by whole exome and transcriptome (RNA-seq) analysis of patient tumor and non-tumor tissues (peripheral blood leukocytes) (n = 15) is shown for each indication. The horizontal line indicates the average TMB within each tumor type. Figure 2B shows preREP yield across multiple indications, as demonstrated by total viable TILs from fresh tumor fragments or single-cell suspensions (n ​​= 31). Each dot represents an individual patient, and the vertical line represents the standard deviation. [Figure 3]Flow cytometry plots of TILs stained for CD134 and CD137 after coculture with unpulsed APCs or APCs pulsed with a pool of 190 peptides containing predicted mutations are shown. Sorting gates are outlined by solid lines or hash marks. TILs expressing CD134 and / or CD137 were sorted and enriched as the selected TIL fraction after coculture with peptide-loaded APCs (3.93%, right panel). Sorting gates were set on live lymphocytes expressing CD4 and / or CD8 before defining the CD134 and CD137 gates. The percentage of CD4+ or CD8+ cells within the sorting gate is indicated in blue in the upper right corner of each flow plot. [Figure 4] Figure 4A shows the fold expansion of sorted T populations (CD134 and / or CD137 selected TILs) at days 10 and 14 of REP. Fold changes were calculated by dividing the total number of viable cells on days 1, 10, and 14 by the number of viable cells seeded on day 1. Figure 4B shows the expansion of selected TILs after 14 days of REP (n=8). Each dot represents an individual patient, and the vertical lines represent the standard deviation. [Figure 5] The phenotype of selected TILs is shown. Figure 5 (left panel) shows the % of CD3+ T cell subsets defined by CD4 and CD8 expression at the end of REP in bulk and selected TILs from CRC (n=3), melanoma (n=1), and ovary (n=1). Figure 5 (right panel) shows T cell memory populations defined based on CD45RA and CCR7 expression within CD4 and CD8 subsets. Each circle represents an individual patient. [Figure 6A] Stacked box plots showing the frequency of TCR clonotypes in selected TILs, bystander cells (negative fraction, unselected), and bulk TILs as determined by single-cell TCR sequencing are shown. The top 18 most frequent clonotypes are indicated by solid boxes, and all other TCR clonotypes identified by scRNA-seq for each sample are shown as single hashed bars. [Figure 6B] Figure 6B shows a decrease in TCR diversity in selected TILs compared to bulk TILs derived from ovarian tumors at the end of REP. Figure 6B (left panel) shows the diversity of TCR clonotypes as assessed by single-cell RNA sequencing for bulk TILs, selected TILs, and negatively selected TILs. Figure 6B (right panel) shows the abundance of TCR clonotypes as assessed by single-cell RNA sequencing for bulk TILs, selected TILs, and negatively selected TILs. Each color block represents a unique TCR, and colored lines connecting samples indicate shared TCR clonotypes. The frequencies of the top 40 most abundant clonotypes across all samples are shown. [Figure 7A] Shown is CD134+CD137+ expression in bulk and selected TILs from CRC (n=3) and melanoma (n=1) as assessed by co-culturing selected and bulk TILs at the end of REP with peptide-pulsed or non-pulsed APCs at a 5:1 ratio. [Figure 7B] Intracellular IFN-γ production in CD4 or CD8 T cells from bulk TILs (far left for each condition), selected TILs (middle bar for each condition), and negative fraction bystander cells (far right for each condition) after coculture with unloaded (DMSO) or peptide-loaded APCs (B cells) at a 5:1 effector:target ratio is shown. Error bars represent the standard error of the mean for triplicate wells. ****P<0.0001, two-way ANOVA. [Figure 7C] Figure 7C (left panel) shows enrichment for neoantigen (i.e., neoantigen peptide) reactivity of selected TILs compared to bulk TILs after REP, as assessed by T cell activation (CD134+CD137+) and intracellular cytokine production of IFN-γ. Figure 7C (right panel) shows enrichment for neoantigen reactivity as indicated by the fold change in CD134+CD137+ in selected TILs compared to bulk TILs. [Figure 8A] Cytokine secretion is shown for bulk TILs (far left for each condition), selected TILs (middle bar for each condition), and negative fraction bystander cells (far right for each condition) after 24 hours of coculture with unloaded (DMSO) or peptide-loaded APCs (B cells) at a 5:1 effector:target (E:T) ratio. Error bars represent the standard error of the mean for triplicate wells. *****P<0.0001, two-way ANOVA. [Figure 8B] Figure 1 shows increased cytokine secretion from selected TILs compared to bulk TILs in response to neoantigen (i.e., neoantigen peptide)-specific stimulation as quantified by multiplex cytometric bead array for IFN-γ and TNFα levels in supernatants collected 24 hours after coculture with peptide-pulsed or non-pulsed APCs. Dots represent technical replicates, and error bars indicate standard deviation of triplicates. [Figure 9-1] Figure 9A shows CD107a expression in bulk TILs (far left for each condition), selected TILs (middle bar for each condition), and negative fraction bystander cells (far right for each condition) after coculture with unloaded (DMSO) or peptide-loaded APCs (B cells) at a 5:1 effector:target ratio. Error bars indicate the standard error of the mean for triplicate wells. ****P<0.0001, two-way ANOVA. Figure 9B shows increased CD107a expression in the CD4+ and CD8+ subsets of selected TILs compared to bulk TILs in response to neoantigen (i.e., neoantigen peptide)-specific stimulation after coculture with peptide-pulsed or unpulsed APCs. Error bars indicate the standard deviation for triplicates. [Figure 9-2]Figure 9C shows granzyme B expression in CD8 T cells of bulk TILs (far left for each condition), selected TILs (middle bar for each condition), and negative fraction bystander cells (far right for each condition) after coculture with unloaded (DMSO) or peptide-loaded APCs (B cells) at a 5:1 effector:target ratio. Error bars represent the standard error of the mean for triplicate wells. ***P<0.0005, two-way ANOVA. Figure 9D shows increased granzyme B expression in the CD8+ subset of selected TILs compared to bulk TILs in response to neoantigen (i.e., neoantigen peptide)-specific stimulation after coculture with peptide-pulsed or unpulsed APCs. Dots represent technical replicates, and error bars indicate the standard deviation of triplicates. [Figure 10] Figures 10A-10B show that selected TILs are functional in response to nonspecific polyclonal stimulation. Bulk and selected TILs from CRC (n = 3) and melanoma (n = 1) were stimulated overnight with soluble CD3 / CD28 activators (CD134+CD137+) or for 5 hours with PMA / ionomycin (IFN-γ, TNFα, and CD107a), and the cells were assessed by flow cytometry for expression of CD134+CD137+, IFN-γ, TNFα, and CD107a on (Figure 10A) CD4+ cells and (Figure 10B) CD8+ cells. Each circle represents an individual sample, with bulk TILs annotated by open circles and selected TILs annotated by filled circles. Horizontal lines indicate the mean percent expression for each population. [Figure 11] Figures 11A-11B show the results of deconvolution analysis of peptide reactivity as determined by CD4+ T cells (Figure 8A) and CD8+ T cells (Figure 8B) in selected TILs that were IFN-γ+ after co-culture of TILs with unloaded APCs or APCs loaded with a smart peptide pool of 190 peptides or 13-14 peptides. [Figure 12] 1 shows an exemplary process for generating TILs enriched for reactivity against mutations expressed in neoantigens from patient material. [Figure 13] TIL expansion from digested colorectal cancer (CRC) and gastric tumor samples is shown in FIG. [Figure 14A] Shown is the upregulation of CD134 (OX40) / CD137 (4-1BB) and the expression of IFNγ, TNFα and granzyme B in the cell supernatant. [Figure 14B] An exemplary gating strategy for sorting neoantigenic peptide-reactive (+ / +) and neoantigenic peptide-unreactive (- / -) TILs is shown in Figure 14B. [Figure 14C] The fold increase of selected samples after the Rapid Expansion Protocol (REP) is shown. [Figure 15A] Representative samples of neoantigen-reactive (+ / +) and non-reactive (- / -) TILs after REP are shown in Figure 15A. [Figure 15B] Reactivity is shown in Figure 15B. Reactivity was measured by upregulation of CD137 (4-1BB) and CD134 (OX40) after co-culture. [Figure 15C] Reactivity is shown as measured by upregulation of granzyme B, IFNγ and TNFα secretion in the supernatant in an ELLA assay. [Figure 15D] Peptides were further screened for individual reactivity by upregulating IFNγ secretion, as shown in FIG. 15D. DETAILED DESCRIPTION OF THE INVENTION

[0041] Detailed Description Various aspects of the present invention provide compositions of tumor-infiltrating lymphocytes (TILs) enriched for tumor-reactive cells, wherein the cells are characterized by TCR clonal diversity and exhibit robust neoantigen reactivity to neoantigenic peptides, particularly compared to TIL compositions with less TCR clonal diversity. Related aspects provide methods for producing such tumor-reactive cell-enriched tumor-infiltrating lymphocytes (TILs).

[0042] The provided compositions and methods relate to the generation of T cell therapies that respond to tumor-associated antigens, such as neoantigens (i.e., neoantigenic peptides). 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 neoantigenic peptides, 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+ and CD4+ T cells, respectively.

[0043] In certain embodiments of the compositions and methods provided, the tumor-reactive cells express a T cell receptor (TCR) that can recognize a neo-antigen (ie, a neo-antigenic peptide).

[0044] 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.

[0045] In some aspects, the reactive T cells are tumor-reactive T cells that recognize cancer neoantigens (i.e., neoantigen peptides). Most neoantigens arise from passenger mutations, which means they do not confer any growth advantage to cancer cells. A small number of mutations actively promote tumor growth, and these are known as driver mutations. Passenger mutations are likely to give rise to neoantigens that are unique to each patient and may be present in every subset of cancer cells. Driver mutations give rise to neoantigens that are likely to be present in every tumor cell of an individual and potentially shared. In some embodiments of the provided methods, the T cell population contains tumor-reactive T cells that can recognize neoantigens containing passenger mutations and / or driver mutations.

[0046] In some respects, neoantigens (i.e., neoantigenic peptides) are ideal targets for immunotherapy because they are disease-specific. For example, such antigens are not commonly present in the body before cancer develops, making them truly cancer-specific, not expressed on normal cells, and 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 the target antigen 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 resulting from 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.

[0047] Recent clinical trials have demonstrated that T cells isolated from surgically resected tumors possess TCRs that recognize neoantigens (i.e., neoantigenic peptides), and expanding these neoantigen-reactive TIL populations and reinfusing them into patients can sometimes result in dramatic clinical benefit. This personalized therapy has resulted in remarkable clinical responses in selected patients with common epithelial tumors.

[0048] TIL therapy has proven most effective in melanoma. Furthermore, enhancing tumor reactivity through selective expansion of individual TIL subpopulations screened for neoantigen reactivity has demonstrated some success in breast cancer (Zacharakis et al., 2022, J. Clin. Oncol. JCO2102170). However, while other solid tumors, such as colorectal cancer (CRC), have been shown to contain neoantigen-reactive TILs (Parkhurst et al., 2019 Cancer Discov. 9:1022-1035), the ability to selectively enrich these cells has been challenging. Herein, we demonstrate the generation of TIL compositions with improved tumor reactivity for solid tumors, such as CRC. In some aspects, provided embodiments demonstrate enrichment of tumor-reactive cells through an ex vivo process that utilizes tumor-specific mutation-containing peptides to select neoantigen-reactive TILs by fluorescence-activated single cell sorting (FACS). Such compositions, when generated from tumors of CRC patients, can result in the production of selected TILs that target neoantigens in CRC.

[0049] 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 for desired neoantigens (i.e., neoantigenic peptides) 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 do not respond 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 are CD4 T cells that can limit the reactivity of T cell products. + These additional approaches to expand tumor-reactive T cells ex vivo are not selective, and as a result, non-reactive T cells in the culture may be preferentially expanded over reactive T cells, resulting in an end product lacking satisfactory reactivity and / or with insufficient numbers of tumor-reactive T cells. Methods for generating tumor-reactive T cells for therapy are needed.

[0050] Various embodiments of methods for generating tumor-infiltrating lymphocytes (TILs) enriched for such tumor-reactive cells include ex vivo expansion and generation of T cell therapeutic compositions for use in connection with the treatment of cancers such as melanoma, colorectal cancer (CRC), breast, and liver cancer, among others. In some embodiments, the manufacturing methods involve the propagation and manipulation of patient cells outside the body. In particular embodiments, the methods relate to methods for expanding T cells containing endogenous TCRs specific for tumor-associated antigens (hereinafter "tumor-reactive T cells"). For purposes of this disclosure, reference to tumor-reactive T cells includes T cells that exhibit reactivity to tumor antigens, as evidenced by the ability of TILs to produce or secrete IFNγ or TNFα after exposure to APCs presenting neoantigenic peptides, or to express factors involved in or associated with cytotoxic killing activity (e.g., granzyme B or degranulation factors such as CD107). In some aspects, the frequency of these cells may be low, and expanding these cells to therapeutic doses requires ex vivo methods of enrichment and expansion, as provided by this embodiment.

[0051] Thus, among the embodiments of the provided TIL compositions are those that exhibit substantial tumor-reactive activity, including degranulation and the ability to express greater amounts of IFN-γ and TNF-α in response to APCs (e.g., DCs or B cells) that present neoantigenic peptides. This functional activity is highly preserved even after cryopreservation and thawing of the TIL compositions. The significant increase in cytolytic enzymes and a more robust activation phenotype support the enhanced ability of the expanded TIL compositions to induce apoptosis of tumor targets. The significant tumor reactivity also supports the usefulness of the provided TIL compositions as a therapy.

[0052] The provided methods result in a product containing tumor-reactive T cells that are enriched for multiclonal TCR TIL populations that can target multiple mutations and / or react to a variety of tumor antigens. In some embodiments, the multiclonal TCR TIL population is an oligoclonal TCR TIL population. Thus, the provided methods result in a product containing tumor-reactive T cells that are enriched for multiclonal TCR TIL populations that can target multiple mutations and / or react to a variety of tumor antigens. As such, such tumor-reactive T cells offer advantages over existing methods in which cells are transduced to express a single neoepitope-reactive TCR, or TILs are expanded in bulk, or exhibit limited multiclonality and / or oligoclonality.

[0053] In some embodiments of the provided methods, a source of potential tumor peptides is used to identify TCRs reactive to neoantigens in a process that includes expanding T cells reactive to tumor neoantigen peptides. The provided methods include an ex vivo co-culture method in which a population of T cells present in or expanded from a tumor sample is incubated in the presence of antigen-presenting cells that have been contacted with or presented with 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.

[0054] Figure 1 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 the identification and generation of peptides for use in co-culture with peptide-presenting antigen-presenting cells (APCs) and with autoantigen-specific T cells obtained from the same subject. In some cases, a suspension of dissociated tumor cells containing T cells is obtained or provided from the patient and subjected to an initial pre-expansion with recombinant IL-2 to expand T cells from the tumor, followed by co-culture with antigen-presenting cells loaded with a pool of 20-mer to 40-mer peptides identified from neoantigens and manufactured for presentation on MHC class I and / or MHC class II molecules. After co-culture, tumor-reactive T cells are selected for surface-positive cells for CD134 and / or CD137 by sorting using fluorescence-activated cell sorting (FACs), thereby eliminating potential bystander cells. The selected and sorted cells are then subjected to a rapid expansion protocol using irradiated peripheral blood mononuclear cells (iPBMCs), an agonistic anti-CD3 antibody (e.g., OKT3), and recombinant IL-2 until a threshold number of cells is obtained, typically for 12 to 16 days, e.g., 14 days or about 14 days. Once the 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. In some aspects, this enriched TIL product is contemplated to have superior reactivity compared to a similar TIL product prepared by bulk methods.

[0055] This process can be carried out in the presence of a serum-free medium containing nutrients. One or more or all of the steps can be carried out in a closed system, e.g., without exposing the cells to the outside environment.

[0056] The provided process enriches the initial subpopulation of tumor-reactive T cells expanded from a tumor for cells that are tumor-reactive before the subsequent second expansion step, thereby facilitating the preservation and expansion of cells of interest and limiting the expansion of bystander T cells, which may include cells that are unresponsive to tumor antigens and / or exhibit inhibitory activity. The provided method maximizes the number of tumor-reactive cells that can be collected by co-culturing all cells expanded after the first expansion with peptide-presenting APCs and then selecting cells positive for CD134 and / or CD137 from the total bulk cells after co-culture before the subsequent second expansion. As demonstrated herein, the provided method can result in a 1000-fold or greater expansion of selected tumor-reactive cells using REP, and such cells exhibit robust neoantigen reactivity as demonstrated by cytokine (e.g., IFNγ) production and secretion and production of factors required for cytotoxic killing (e.g., CD107a and granzyme B).

[0057] This is in contrast to existing methods involving passive expansion of bulk T cells, in which all tumor-derived T cells are subjected to expansion. While these alternative processes can significantly expand total viable cells (TVC), these other methods do not actively ensure that tumor-reactive T cells are primarily expanded.

[0058] In certain aspects, the provided methods can be used for ex vivo production of T cell therapy, including ex vivo expansion of autologous tumor-reactive T cells. In some embodiments, the methods generate or expand T cells for use in autologous cell therapy to treat cancer. In certain embodiments, the tumor from which the cells are derived is from colorectal cancer (CRC), and the methods are used to treat CRC in a patient.

[0059] Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0060] All publications referenced in this application, including patent documents, scientific papers, and databases, are incorporated by reference in their entirety for all purposes, 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, patent application, published patent application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0061] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0062] I. Enriched Tumor-Reactive TIL Compositions Provided herein is a TIL composition enriched for tumor-reactive T cells. In some embodiments, the TIL composition contains primary T cells derived from a tumor of a subject, which are enriched for tumor-reactive T cells by co-culture with autologous APCs that present neo-antigen peptides derived from the tumor of the subject and are expanded ex vivo. In some embodiments, the provided TIL composition can be produced by the provided ex vivo method for producing a TIL composition.

[0063] In some embodiments, the provided TIL compositions are multiclonal populations exhibiting TCR diversity and enrichment of T cell receptors (TCRs) that respond to neoantigens (i.e., neoantigenic peptides). In some embodiments, the multiclonal populations are oligoclonal populations exhibiting TCR diversity and enrichment of various TCR clonotypes that respond to neoantigens. In some embodiments, the TIL compositions contain at least 10 different TCR clonotypes that exhibit neoantigen reactivity. In some embodiments, the TIL compositions contain at least 11, 12, 12, 14, 15, 16, 17, 18, 19, 20, or more different TCR clonotypes that exhibit neoantigen reactivity. In some embodiments, the TIL compositions contain more than 20 different TCR clonotypes, e.g., 20-100 different TCR clonotypes, e.g., 20, 30, 40, 50, 60, 70, 80, 90, or 100 different TCR clonotypes, or any value in between any of the foregoing. In some embodiments, the TIL composition contains 30-80 different TCR clonotypes, hi some embodiments, the TIL composition contains 40-60 different TCR clonotypes.

[0064] In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 10 different TCR clonotypes, each having a frequency of at least 2.0% in the population. In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 11 different TCR clonotypes, each having a frequency of at least 2.0% in the population. In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 12 different TCR clonotypes, each having a frequency of at least 2.0% in the population. In some embodiments, 8-15 different T cell receptor (TCR) clonotypes comprise at least 50% of the TCR frequencies in the population. In some embodiments, 9-12 different TCR clonotypes comprise at least 50% of the TCR frequencies in the population.

[0065] In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 10 different TCR clonotypes, each having a frequency of at least 1.0% in the population. In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 11 different TCR clonotypes, each having a frequency of at least 1.0% in the population. In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 12 different TCR clonotypes, each having a frequency of at least 1.0% in the population. In some embodiments, 8-15 different T cell receptor (TCR) clonotypes comprise at least 50% of the TCR frequency in the population. In some embodiments, 9-12 different TCR clonotypes comprise at least 50% of the TCR frequency in the population.

[0066] In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 20 different TCR clonotypes, each having a frequency of at least 2.0% in the population. In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 25 different TCR clonotypes, each having a frequency of at least 2.0% in the population. In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 30 different TCR clonotypes, each having a frequency of at least 2.0% in the population. In some embodiments, the population is enriched for tumor-reactive T cells and comprises at least 40 different TCR clonotypes, each having a frequency of at least 2.0% in the population. In some embodiments, 8-15 different T cell receptor (TCR) clonotypes comprise at least 50% of the TCR frequency in the population. In some embodiments, 9-12 different TCR clonotypes comprise at least 50% of the TCR frequency in the population.

[0067] In some embodiments, the top 40 TCR clonotypes comprise at least 75% of the TCR frequency in a population. In some embodiments, the top 40 TCR clonotypes comprise at least 80% of the TCR frequency in a population. In some embodiments, the top 40 TCR clonotypes comprise at least 85% of the TCR frequency in a population. In some embodiments, the top 40 TCR clonotypes comprise at least 90% of the TCR frequency in a population.

[0068] In some embodiments, the neoantigen reactivity of a TCR clonotype is to at least one CD4 antigen and at least one CD8 antigen, hi some embodiments, the neoantigen reactivity of a TCR clonotype is to at least two peptide antigens, where at least one peptide antigen is a CD4 antigen and at least one peptide antigen is a CD8 antigen.

[0069] In some embodiments, the neoantigen reactivity of a TCR clonotype is to two to six different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to two to four different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to two different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to two different peptide antigens, one CD8 antigen and one CD4 antigen.

[0070] In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-100 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-80 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-60 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-40 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-20 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-10 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-6 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 2-4 different peptide antigens.

[0071] In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 4 to 100 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 4 to 80 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 4 to 60 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 4 to 40 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 4 to 20 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 4 to 10 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to 4 to 6 different peptide antigens.

[0072] In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to between 6 and 100 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to between 6 and 80 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to between 6 and 60 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to between 6 and 40 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to between 6 and 20 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is directed to between 6 and 10 different peptide antigens.

[0073] In some embodiments, the neoantigen reactivity of a TCR clonotype is to 2, 4, 6, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 2 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 4 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 6 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 8 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 10 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 20 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 40 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 60 different peptide antigens. In some embodiments, the neoantigen reactivity of a TCR clonotype is to 80 different peptide antigens, hi some embodiments, the neoantigen reactivity of a TCR clonotype is to 100 different peptide antigens.

[0074] Various methods for evaluating TCR repertoires for clonotype identification and TCR repertoire analysis are known (see, e.g., Rosati et al. (2017) BMC Biotechnology, 17:61; Friedensohn et al. (2016) Trends in Biotechnology, 35:203-214). In some aspects, the method comprises a high-throughput sequencing method or a next-generation sequencing method. In some embodiments, the frequency and diversity of various clones present in a population or composition can be determined. In some embodiments, the composition can be evaluated for clonality, clonal diversity, or clonal heterogeneity of cells within the population of cells, for example, based on the determined frequency and / or diversity of clonotypes present in the population or composition. In some embodiments, single-cell sequencing is performed to identify clonotypes on specific cells. In certain aspects, a paired αβ TCR sequencing method is used (see, e.g., WO2017053902). In some embodiments, sequencing is performed on DNA, such as genomic DNA or complementary DNA.In some embodiments, sequencing is performed on RNA.In some embodiments, high-throughput sequencing or next-generation sequencing of TCR sequence, or whole genome or transcriptome sequencing (such as RNA sequencing) is used.In some aspects, the method used is based on RNA sequencing.

[0075] In some embodiments, T cell clonotype assessment, and clonality and diversity in various T cell populations or compositions or samples containing T cells, are determined for populations or compositions of cells and / or in single cells using high-throughput sequencing of all or a portion of the TCR genes or based on sequences obtained from high-throughput whole genome or transcriptome analysis. In some embodiments, the provided methods can include various features of the methods described in WO 2016 / 044227, WO 2016 / 176322, WO 2012 / 048340, WO 2012 / 048341, WO 2014 / 144495, WO 2017 / 053902, WO 2017 / 053903, or WO 2017 / 053905, each of which is incorporated by reference in its entirety.

[0076] In some examples, the clonotype of a cell, or the clonotype present in a cell population or composition, can be determined by TCR sequencing. In some embodiments, the sequencing method that can be used includes high-throughput sequencing or next-generation sequencing known in the art. In some aspects, next-generation sequencing methods using genomic DNA or cDNA from T cells can be used to evaluate the TCR repertoire, including the sequence encoding complementarity-determining region 3 (CDR3). In some embodiments, whole transcriptome sequencing by RNA sequencing can be used. In some aspects, whole transcriptome sequencing (e.g., RNA sequencing) can be used to construct or extract TCR repertoire information, such as TCR sequence and relative frequency. For example, in some aspects, computational methods such as MIXCR (such as those described in Bolotin et al. Nature Methods 12 (2015) 380-381, Bolotin et al., Nature Biotechnology 35 (2017) 908-911) or IMREP (Mangul et al., bioRxiv (2017) 089235) can be used to determine repertoire TCR sequences or portions thereof (e.g., CDR3) from whole-transcriptome RNA-seq results. In some embodiments, single-cell sequencing methods can be used. In some embodiments, clonotypes can be assessed or determined by spectratype analysis (a measure of TCR Vβ, Vα, Vγ, or Vδ chain hypervariable region repertoires). Clonotypes can also be determined by generating and characterizing antigen-specific clones against an antigen of interest.

[0077] In some embodiments, T cell clonotype assessment is determined for a population or composition of cells and / or in single cells using high-throughput sequencing of all or part of the TCR gene, or based on sequences obtained from high-throughput whole genome or transcriptome analysis. In some embodiments, bulk sequencing of target sequences (e.g., TCR chains or parts thereof), or bulk whole genome or transcriptome sequencing (e.g., by RNA sequencing) can be used to determine the clonotypes present in cells in a population or composition. In some aspects, T cell clonotype assessment can include sequencing of a portion of the variable region of one or more native TCR chains, such as complementarity-determining region 3 (CDR3). In some aspects, single-cell sequencing can be used. In some embodiments, the provided methods can include various features of the methods described in WO 2016 / 044227, WO 2016 / 176322, WO 2012 / 048340, WO 2012 / 048341, WO 2014 / 144495, WO 2017 / 053902, WO 2017 / 053903, or WO 2017 / 053905, each of which is incorporated by reference in its entirety. In some embodiments, for the target TCR molecule, the genes encoding the chains of the TCR can be obtained from genomic DNA or mRNA of immune cells or T cells.

[0078] In some embodiments, the composition exhibits clonal diversity, i.e., is multiclonal, such as oligoclonal. In some cases, clonal diversity is determined based on the relative frequency of one or more clonotypes and / or one or more TCR sequences. For example, oligoclonality or grammatical variations thereof refers to clonotypes derived from a small number of clones. The relative number of specificities for determining whether a multiclonal population is oligoclonal is not necessarily a defined number, generally 10 or fewer antigen specificities, but may be higher or lower. In some embodiments, an oligoclonal composition has 2 to 10 different specificities. In some embodiments, determining clonal diversity is expressed as the clonality, Shannon-adjusted clonality, or top 25 clonality of each of a plurality of samples. In some embodiments, determining clonal diversity is expressed as the Shannon-adjusted clonality of the composition.

[0079] In some embodiments, the cells of the provided TIL compositions exhibit one or more phenotypic or functional markers. In some cases, such cells include cells that are positive or negative for one or more phenotypic markers or functional characteristics or attributes.

[0080] As used herein, the statement that a cell or cell population is " positive " for a certain marker, function or attribute refers to the presence of a specific marker, such as a surface marker, that can be detected on or in the cell.When referring to a surface marker, this term refers to the presence of surface expression, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, detected by flow cytometry, and the staining is substantially higher than the staining detected by carrying out the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially similar to that for cells known to be positive for the marker, and / or at a level substantially higher than that for cells known to be negative for the marker.

[0081] As used herein, the statement that a cell or cell population is " negative " for a specific marker, function or attribute refers to the fact that a specific marker, for example, a surface marker, is not substantially detectably present on or in cells.When referring to a surface marker, this term refers to the absence of surface expression, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody, detected by flow cytometry, and the staining is not detected by flow cytometry at a level substantially higher than the staining detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than the level for cells known to be positive for the marker, and / or at a level substantially similar to the level for cells known to be negative for the marker.

[0082] Exemplary markers, functions, and attributes of the provided TIL compositions are described below. In some embodiments, the TIL compositions are characterized by any one or more of these characteristics, for example, two, three, four, five, or more of these characteristics. For example, the provided TIL compositions can be characterized by the presence or absence of one or more T cell markers, effector memory phenotype markers, exhaustion markers, the ability to produce or secrete cytokines, and / or the ability to produce or secrete cytotoxic factors, for example, as described below. Any two, three, four, five, or more of these characteristics can be present in the described TIL compositions.

[0083] In some embodiments, the provided TIL compositions comprise CD3+ T cells as a percentage of total cells in the population that is greater than or about 85%, e.g., greater than or about 90%, greater than or about 90%, greater than or about 95%, greater than or about 95%, greater than or about 97%, or greater than or about 97%, or greater than or about 98%. In some embodiments, the provided TIL compositions comprise CD3+ T cells as a percentage of total cells in the population that is greater than or about 90%. In some embodiments, the provided TIL compositions comprise CD3+ T cells as a percentage of total cells in the population that is greater than or about 95%. In some embodiments, the provided TIL compositions comprise CD3+ T cells as a percentage of total cells in the population that is greater than or about 98%. In some embodiments, the composition contains CD4+ T cells and CD8+ T cells as a percentage of total cells in the population that is greater than or about 85%, greater than or about 90%, greater than or about 90%, greater than or about 95%, greater than or about 97%, or greater than or about 97%, or greater than or about 98%.

[0084] In some embodiments, the composition has a glycerol ratio of 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:1, or about 5:1 to 50:1, The composition comprises a ratio of CD4+ T cells to CD8+ T cells of about 5:1 to 25:1, about 5:1 to 20:1, about 5:1 to 15:1, about 5:1 to 10:1, about 10:1 to 50:1, about 10:1 to 25:1, about 10:1 to 20:1, about 10:1 to 15:1, about 15:1 to 50:1, about 15:1 to 25:1, about 15:1 to 20:1, about 20:1 to 50:1, about 20:1 to 25:1, or about 25:1 to 50:1. In some embodiments, the composition comprises a ratio of CD4+ T cells to CD8+ T cells of 10:1 to 25:1 or about 10:1 to 25:1. In some embodiments, the composition comprises a ratio of CD4+ T cells to CD8+ T cells of about 20:1.

[0085] In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 50% express markers of the effector memory phenotype. In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 50% express effector memory phenotype markers, greater than about 60% express effector memory phenotype markers, greater than about 70% express effector memory phenotype markers, greater than about 80% express effector memory phenotype markers, or greater than 90% express effector memory phenotype markers. In some embodiments, the effector memory phenotype is characterized by the expression of CD45RA-, CD45RO+, CD62L -In some embodiments, the effector memory phenotype is characterized by surface marker expression that is one or more of CD45RA- and CCR7-. In some embodiments, the effector memory phenotype is characterized by surface marker expression that is one or more of CD45RA-CD45RO+, CD62L - , and CCR7 - In some embodiments, the effector memory phenotype is characterized by surface marker expression of CD45RA-, CD45RO + , CD62L - , CCR7 - , CD28 - and CD27.

[0086] In some embodiments, among the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, more than about 50% are CD45RA- and CCR7-, more than about 60% are CD45RA- and CCR7-, more than about 70% are CD45RA- and CCR7-, more than about 80% are CD45RA- and CCR7-, or more than 90% are CD45RA- and CCR7-.

[0087] In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, more than about 10% express a central memory T cell marker, or more than about 15% express a central memory T cell marker, or more than about 20% express a central memory T cell marker, or more than 25% express a central memory T cell marker. In some embodiments, the central memory T cell marker is CD45RA-CCR7+. In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, more than about 10% are CD45RA-CCR7+, or more than about 15% are CD45RA-CCR7+, or more than about 20% are CD45RA-CCR7+, or more than 25% are CD45RA-CCR7+.

[0088] In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 10% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 15% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 20% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 25% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-).

[0089] In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 10% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 15% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 20% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 25% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-).

[0090] In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 10% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 80% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than about 15% express a central memory T cell marker (e.g., CD45RA-CCR7+) and greater than about 80% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, among the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, about 20% express central memory T cell markers (e.g., CD45RA-CCR7+) and about 80% express effector memory phenotype markers (e.g., CD45RA-CCR7-).

[0091] In some embodiments, among the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, about 10% express central memory T cell markers (e.g., CD45RA-CCR7+) and about 90% express effector memory phenotype markers (e.g., CD45RA-CCR7-).

[0092] In some embodiments, among the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, less than 30% of the cells express an exhausted phenotype. In some embodiments, among the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, less than about 25% express an exhausted phenotype, less than about 20% express an exhausted phenotype, less than about 15% express an exhausted phenotype, or less than about 10% express an exhausted phenotype. In some cases, exhaustion can be assessed by monitoring the loss of T cell function, for example, a decrease or reduction in neoantigen-specific reactivity, for example, a decrease or reduction in the ability to produce cytokines or to drive cytolytic activity against target antigens. In some cases, exhaustion can also be assessed by monitoring the expression of surface markers on T cells (e.g., CD3+ T cells, or their CD4 and / or CD4+ T cell subsets) associated with an exhausted phenotype. Among the exhaustion markers are inhibitory receptors such as PD-1, CTLA-4, LAG-3, and TIM-3. In some embodiments, the exhaustion phenotype is the positive expression of one, two, three, or four of such exhaustion markers.

[0093] In some embodiments, among the CD3+ T cells in the TIL composition, or the CD4+ and / or CD8+ T cell subsets thereof, greater than 70% are PD-1 - In some embodiments, among the CD3+ T cells in the TIL composition, or the CD4+ and / or CD8+ T cell subsets thereof, greater than 75% express PD-1 - or >80% PD-1 - or >85% PD-1 - or >90% PD-1 - or >95% PD-1 - is.

[0094] In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 70% are LAG3 -In some embodiments, among the CD3+ T cells in the TIL composition, or CD4+ and / or CD8+ T cell subsets thereof, greater than 75% are LAG3 - or >80% LAG3 - or >85% LAG3 - or >90% LAG3 - or >95% LAG3 - is.

[0095] In some embodiments, among the CD3+ T cells in the TIL composition, or the CD4+ and / or CD8+ T cell subsets thereof, greater than 70% are PD-1 - and LAG3 - In some embodiments, among the CD3+ T cells in the TIL composition, or the CD4+ and / or CD8+ T cell subsets thereof, greater than 75% express PD-1 - and LAG3 - or >80% PD-1 - and LAG3 - or >85% PD-1 - and LAG3 - or >90% PD-1 - and LAG3 -- or >95% PD-1 - and LAG3 - is.

[0096] In some embodiments, the provided TIL compositions comprise about 10-60% tumor-reactive T cells. In some embodiments, the provided TIL compositions comprise greater than about 15% tumor-reactive T cells, greater than about 20% tumor-reactive T cells, greater than about 25% tumor-reactive T cells, greater than about 30% tumor-reactive T cells, greater than about 40% tumor-reactive T cells, or greater than about 50% tumor-reactive T cells, or any value between any of the foregoing.

[0097] The provided TIL compositions enriched for tumor-reactive cells exhibit several functional or phenotypic activities that demonstrate their reactivity to neoantigens (i.e., neoantigenic peptides). In some embodiments, the cells can be evaluated for any of several functional or phenotypic activities, including, but not limited to, cytotoxic activity, degranulation, the ability to produce or secrete cytokines, and the expression of one or more intracellular or surface phenotypic markers. Methods for evaluating such activities are known and are exemplified herein and in the Examples.

[0098] In some embodiments, TILs can be activated upon recognition of neoantigens (i.e., neoantigenic peptides) presented by APCs. Upon activation, TILs abundantly produce cytokines, chemokines, and other factors, and simultaneously exhibit potent cytolytic activity. In some embodiments, activation triggers the release of cytoplasmic granules containing granzymes, resulting in target cell death. Assays for measuring cytokines, chemokines, and other soluble factors are well known in the art and include, but are not limited to, ELISA, intracellular cytokine staining, cytometric bead arrays, RT-PCR, ELISPOT, flow cytometry, and bioassays in which cells responsive to the relevant cytokine are tested for responsiveness (e.g., proliferation) in the presence of a test sample.

[0099] In some embodiments, TILs can be evaluated for phenotype or general functional activity, such as IFN-γ and / or granzyme B secretion or other cytokine secretion, in response to polyclonal stimulation. In some embodiments, polyclonal stimulation is CD3 stimulation (e.g., with OKT3). In some embodiments, in vitro CD3 assays include OKT3 stimulation. In some embodiments, in vitro CD3 assays include washing TILs and seeding them on a culture plate pre-coated with OKT3 diluted in phosphate-buffered saline. In some embodiments, polyclonal stimulation is CD3 (e.g., with OKT3) and CD28 stimulation to provide a costimulatory signal. In some embodiments, in vitro assays include stimulation with anti-CD3 and anti-CD28 antibodies, for example, by incubating cells with Dynabeads. After overnight incubation, the supernatant is collected, and the protein in the supernatant is measured for the cytokine of interest by ELISA.

[0100] In some embodiments, the provided TIL compositions are evaluated for tumor or neoantigen reactivity, for example, by an in vitro assay. In some embodiments, the assay can be an in vitro autologous tumor assay. In some embodiments, the assay is an in vitro co-culture assay.

[0101] In these and related embodiments, results from such assays (e.g., in vitro autologous tumor assays or in vitro co-culture assays or similar assays) can be used as criteria for characterizing the TIL compositions and / or the cell populations comprising the compositions. Such criteria can include, without limitation, the presence and / or amount or level of one more of the following: cytotoxic activity (e.g., tumor cell killing), production and / or secretion of one more of cell activation and / or reactivity (e.g., against tumor cells) cytokines (e.g., IFN-γ and / or granzyme B secretion), or production or secretion of other compounds associated with one or more of cytotoxic activity, cell activation, cell reactivity, cell viability, or cell exhaustion.

[0102] In some embodiments, TILs can be assessed for cytokine secretion, e.g., IFN-γ and / or granzyme B secretion, in response to coculture with autologous tumor digest in an in vitro autologous tumor assay. In some embodiments, reference to an in vitro autologous tumor assay is understood to be an assay in which TILs are incubated with non-hematopoietic cells derived from an autologous primary tumor. In some embodiments, the in vitro autologous tumor assay involves seeding TILs (e.g., at a 1:1 ratio) into culture plates containing autologous non-hematopoietic tumor cells. In some embodiments, the autologous tumor cells are a single-cell suspension of CD45-negative (CD45-) cells obtained from the primary tumor. After an incubation period ranging from 12 to 24 hours, supernatants can be harvested and factor release can be quantified, e.g., by ELISA.

[0103] In some embodiments, TILs can be evaluated for cytokine secretion, e.g., IFN-γ and / or granzyme B secretion, in response to co-culture with APCs bearing neoantigens (i.e., neoantigenic peptides) in an in vitro co-culture assay. In some embodiments, reference to an in vitro co-culture assay is understood to be an assay in which TILs are incubated with autologous APCs bearing autologous neoantigenic peptides (hereinafter also referred to as peptide-bearing autologous APCs). In some embodiments, the in vitro co-culture assay involves seeding TILs into a culture plate containing autologous irradiated APCs presenting the neoantigenic peptides. In some embodiments, the APCs are irradiated. In some embodiments, the APCs are blood-derived APCs, such as B cells or dendritic cells. In some embodiments, the in vitro co-culture assay referred to herein is an assay in which B cells are isolated and expanded from autologous blood or apheresis by culturing them for 14 days, e.g., with CD40L and IL-4, followed by co-culture with TILs at a TIL:APC ratio ranging from 1:1 to 1:5, carrying a neo-antigenic peptide. After an incubation period ranging from 12 to 24 hours, supernatants can be harvested and factor release can be quantified, e.g., by ELISA. In some aspects, in vitro co-culture assays result in robust T cell activation. While not wishing to be bound by theory, it is believed that the APCs present in the in vitro co-culture assay, in addition to being pulsed with the neo-antigenic peptide cognate to the TIL TCR, express robust levels of HLA and costimulatory molecules necessary for optimal T cell activation.

[0104] In some embodiments, the provided TIL compositions comprise an increased or expanded percentage of cells exhibiting neoantigen reactivity compared to bulk TIL compositions. In certain embodiments, reference to bulk TIL compositions refers to a TIL population expanded from the same input tumor cell source as the provided TIL compositions, but which has not been enriched for tumor-reactive cells by co-culture with APCs presenting neoantigenic peptides and selection for cells positive for CD134 and CD137. For example, in some embodiments, bulk TIL compositions refer to a TIL population that has been processed the same or substantially the same as the provided TIL compositions generated as described in Sections II.A-E, except that the entire bulk population of T cells from the tumor sample is not subjected to selection for cells positive for CD134 and CD137, such that the entire bulk population of T cells from the tumor sample is subjected to ex vivo expansion. In some embodiments, neoantigen reactivity is increased by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, more than 9-fold, more than 10-fold, more than 15-fold, more than 20-fold, more than 30-fold, more than 40-fold, more than 50-fold or more.

[0105] In some embodiments, the provided TIL compositions exhibit higher neoantigen reactivity than bulk TIL compositions in neoantigen reactivity assays, such as in vitro co-culture assays or in vitro autologous tumor assays. In some embodiments, among the CD8+ T cells in the provided TIL compositions, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro autologous tumor assay. In some embodiments, among the CD4+ T cells in the provided TIL compositions, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro autologous tumor assay. In some embodiments, of the total T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro autologous tumor assay. In some embodiments, of the total cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro autologous tumor assay. In some embodiments, the TIL composition exhibits greater than about 2-fold, greater than about 3-fold, greater than about 4-fold, or greater than about 5-fold neoantigen reactivity in an in vitro autologous tumor assay compared to the bulk TIL composition.

[0106] In some embodiments, among the CD8+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neoantigen peptide. In some embodiments, among the CD4+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neoantigen peptide. In some embodiments, of the total T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neoantigen peptide. In some embodiments, of the total cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit neoantigen reactivity in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neoantigen peptide. In some embodiments, the TIL composition exhibits about 2-fold greater neoantigen reactivity, about 3-fold greater neoantigen reactivity, about 4-fold greater neoantigen reactivity, or about 5-fold greater neoantigen reactivity in an in vitro coculture assay, for example, after culture with autologous APCs (e.g., DCs or B cells) that present neoantigen peptides, compared to bulk TIL composition.

[0107] In some embodiments, the TIL composition can comprise an increased or expanded percentage of CD3+ T cells positive for CD134 and CD137 compared to the percentage of such CD3+ T cells positive for CD134 and CD137 present in the bulk TIL population, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide in a coculture assay. In some embodiments, the percentage is increased by at least, or at least about, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold or more.

[0108] In some embodiments, greater than or about 48% of the cells in the provided TIL compositions are positive for CD134 and CD137, e.g., greater than or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more. In some embodiments, greater than or about 50% of the cells in the provided TIL compositions are positive for CD134 and CD137. In some embodiments, greater than or about 60% of the cells in the provided TIL compositions are positive for CD134 and CD137. In some embodiments, greater than or about 70% of the cells in the provided TIL compositions are positive for CD134 and CD137. In some embodiments, greater than or greater than about 80% of the cells in the provided TIL compositions are positive for CD134 and CD137.

[0109] In some embodiments, the provided TIL compositions exhibit a higher effector cytokine response than bulk TIL compositions in a neoantigen reactivity assay, such as an in vitro co-culture assay or an in vitro autologous tumor assay. In some embodiments, among the CD8+ T cells in the provided TIL compositions, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce IFN-γ in an in vitro co-culture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neoantigen peptide. In some embodiments, among the CD4+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce IFN-γ in an in vitro co-culture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, among the total T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce IFN-γ in an in vitro co-culture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, of the total cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce IFN-γ in an in vitro co-culture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide.

[0110] In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the CD8+ T cells in a provided TIL composition produce IFN-γ in an in vitro autologous tumor assay. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the CD4+ T cells in a provided TIL composition produce IFN-γ in an in vitro autologous tumor assay. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the total T cells in a provided TIL composition produce IFN-γ in an in vitro autologous tumor assay. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the total cells in a provided TIL composition produce IFN-γ in an in vitro autologous tumor assay.

[0111] In some embodiments, the TIL composition produces about 2-fold more IFN-γ compared to bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 3-fold more IFN-γ, about 4-fold more IFN-γ, about 5-fold more IFN-γ, about 6-fold more IFN-γ, about 7-fold more IFN-γ, about 8-fold more IFN-γ, about 9-fold more IFN-γ, about 10-fold more IFN-γ, or about 15-fold more IFN-γ compared to bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 20-fold more IFN-γ compared to bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 30-fold more IFN-γ, about 40-fold more IFN-γ, about 50-fold more IFN-γ, about 60-fold more IFN-γ, about 70-fold more IFN-γ, about 80-fold more IFN-γ, about 90-fold more IFN-γ, or about 100-fold more IFN-γ compared to bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 300-fold more IFN-γ, about 400-fold more IFN-γ, about 500-fold more IFN-γ, about 600-fold more IFN-γ, about 700-fold more IFN-γ, about 800-fold more IFN-γ, about 900-fold more IFN-γ, or about 1000-fold more IFN-γ in an in vitro coculture assay compared to a bulk TIL composition, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present neo-antigenic peptides.

[0112] In some embodiments, the TIL composition produces about 2-fold more IFN-γ after in vitro autologous tumor assays compared to bulk TIL composition. In some embodiments, the TIL composition produces about 3-fold more IFN-γ, about 4-fold more IFN-γ, about 5-fold more IFN-γ, about 6-fold more IFN-γ, about 7-fold more IFN-γ, about 8-fold more IFN-γ, about 9-fold more IFN-γ, about 10-fold more IFN-γ, or about 15-fold more IFN-γ after in vitro coculture assays, e.g., after culture with autologous APCs (e.g., DCs or B cells) presenting neo-antigenic peptides, compared to bulk TIL composition. In some embodiments, the TIL composition produces about 20-fold more IFN-γ after in vitro autologous tumor assays compared to bulk TIL composition. In some embodiments, the TIL composition produces about 30-fold more IFN-γ, about 40-fold more IFN-γ, about 50-fold more IFN-γ, about 60-fold more IFN-γ, about 70-fold more IFN-γ, about 80-fold more IFN-γ, about 90-fold more IFN-γ, or about 100-fold more IFN-γ in an in vitro autologous tumor assay compared to bulk TIL composition. In some embodiments, the TIL composition produces about 300-fold more IFN-γ, about 400-fold more IFN-γ, about 500-fold more IFN-γ, about 600-fold more IFN-γ, about 700-fold more IFN-γ, about 800-fold more IFN-γ, about 900-fold more IFN-γ, or about 1000-fold more IFN-γ in an in vitro autologous tumor assay compared to bulk TIL composition.

[0113] In some embodiments, the TIL composition produces IFN-γ after in vitro coculture assays, e.g., culture with autologous APCs (e.g., DCs or B cells) presenting neoantigenic peptides. In some embodiments, the TIL composition produces 100-500 pg / mL, 500-1000 pg / mL, 1,000-2,000 pg / mL, or 2,000-2,500 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000-4,000 pg / mL, 2,000-6,000 pg / mL, 2,000-20,000 pg / mL, 2,000-30,000 pg / mL, or 2,000-40,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000-2,500 pg / mL, 2,000-3,000 pg / mL, 2,000-3,500 pg / mL, 2,000-4,000 pg / mL, or 2,000-4,500 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000-1,500,000 pg / mL, 2,000-1,000,000 pg / mL, 2,000-500,000 pg / mL, or 2,000-250,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 5,000-1,500,000 pg / mL, 5,000-1,000,000 pg / mL, 5,000-500,000 pg / mL, or 5,000-250,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 5,000-1,500,000 pg / mL, 100,000-1,000,000 pg / mL, 100,000-500,000 pg / mL, or 100,000-250,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 500 pg / mL, 1,000 pg / mL, 1,500 pg / mL, 2,000 pg / mL, 2,500 pg / mL, or 3,000 pg / mL of IFN-γ, hi some embodiments, the TIL composition produces 2,000 pg / mL, 4,000 pg / mL, 6,000 pg / mL, 8,000 pg / mL, 10,000 pg / mL, or 20,000 pg / mL of IFN-γ.In some embodiments, the TIL composition produces 5,000 pg / mL, 10,000 pg / mL, 20,000 pg / mL, 30,000 pg / mL, 40,000 pg / mL, or 50,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 50,000 pg / mL, 100,000 pg / mL, 200,000 pg / mL, 300,000 pg / mL, 400,000 pg / mL, 500,000 pg / mL, 600,000 pg / mL, 700,000 pg / mL, 800,000 pg / mL, 900,000 pg / mL, 1,000,000 pg / mL, or 1,500,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 100 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 500 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 1,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 4,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 5,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 6,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 20,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 30,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 40,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 50,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 500,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 600,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 700,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 800,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 900,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 1,000,000 pg / mL of IFN-γ.In some embodiments, the TIL composition produces 1,500,000 pg / mL of IFN-γ.

[0114] In some embodiments, the TIL composition produces IFN-γ after an in vitro autologous tumor assay. In some embodiments, the TIL composition produces 100-500 pg / mL, 500-1000 pg / mL, 1,000-2,000 pg / mL, or 2,000-2,500 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000-4,000 pg / mL, 2,000-6,000 pg / mL, 2,000-20,000 pg / mL, 2,000-30,000 pg / mL, or 2,000-40,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000-2,500 pg / mL, 2,000-3,000 pg / mL, 2,000-3,500 pg / mL, 2,000-4,000 pg / mL, or 2,000-4,500 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000-15,000 pg / mL, 2,000-10,000 pg / mL, or 2,000-5,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 3,000-15,000 pg / mL, 3,000-10,000 pg / mL, or 3,000-5,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 5,000-15,000 pg / mL, 5,000-10,000 pg / mL, or 5,000-5,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000 pg / mL, 5,000 pg / mL, 10,000 pg / mL, or 15,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 100 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 500 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 1,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 4,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 5,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 6,000 pg / mL of IFN-γ.In some embodiments, the TIL composition produces 10,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 15,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 20,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 30,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 40,000 pg / mL of IFN-γ.

[0115] In some embodiments, among the CD8+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce TNF-α in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, among the CD4+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce TNF-α in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, of the total T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce TNF-α in an in vitro co-culture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, of the total cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% produce TNF-α in an in vitro co-culture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide.

[0116] In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the CD8+ T cells in a provided TIL composition produce TNF-α in an in vitro autologous tumor assay. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the CD4+ T cells in a provided TIL composition produce TNF-α in an in vitro autologous tumor assay. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the total T cells in a provided TIL composition produce TNF-α in an in vitro autologous tumor assay. In some embodiments, greater than or about 15%, greater than or about 20%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% of the total cells in a provided TIL composition produce TNF-α in an in vitro autologous tumor assay.

[0117] In some embodiments, the TIL composition produces about 50-fold more TNF-α compared to bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 2-fold more, about 5-fold more, about 10-fold more, about 15-fold more, about 20-fold more, about 25-fold more, about 30-fold more, about 35-fold more, or about 40-fold more TNF-α compared to bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 75 times more TNF-α, about 100 times more TNF-α, about 150 times more TNF-α, about 200 times more TNF-α, about 250 times more TNF-α, about 300 times more TNF-α, about 350 times more TNF-α, or about 400 times more TNF-α in an in vitro coculture assay compared to a bulk TIL composition, for example, after culture with autologous APCs (e.g., DCs or B cells) that present neo-antigenic peptides.

[0118] In some embodiments, the TIL composition produces about 50-fold more TNF-α after an in vitro autologous tumor assay compared to a bulk TIL composition. In some embodiments, the TIL composition produces about 2-fold more TNF-α, about 5-fold more TNF-α, about 10-fold more TNF-α, about 15-fold more TNF-α, about 20-fold more TNF-α, about 25-fold more TNF-α, about 30-fold more TNF-α, about 35-fold more TNF-α, or about 40-fold more TNF-α after an in vitro autologous tumor assay compared to a bulk TIL composition. In some embodiments, the TIL composition produces about 75 times more TNF-α, about 100 times more TNF-α, about 150 times more TNF-α, about 200 times more TNF-α, about 250 times more TNF-α, about 300 times more TNF-α, about 350 times more TNF-α, or about 400 times more TNF-α in an in vitro autologous tumor assay compared to a bulk TIL composition.

[0119] In some embodiments, the TIL composition produces TNF-α in an in vitro coculture assay, e.g., in culture with autologous APCs (e.g., DCs or B cells) presenting neo-antigenic peptides. In some embodiments, the TIL composition produces 50-100 pg / mL, 50-150 pg / mL, 50-200 pg / mL, 50-400 pg / mL, 50-500 pg / mL, 50-600 pg / mL, 50-700 pg / mL, 50-800 pg / mL, 50-900 pg / mL, or 50-1000 pg / mL of TNF-α. In some embodiments, the TIL composition produces 250-2500 pg / mL, 250-2000 pg / mL, 250-1500 pg / mL, 250-1000 pg / mL, or 250-500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 250 pg / mL, 500 pg / mL, 1000 pg / mL, 1500 pg / mL, 2000 pg / mL, or 2500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 50 pg / mL of TNF-α. In some embodiments, the TIL composition produces 100 pg / mL of TNF-α. In some embodiments, the TIL composition produces 200 pg / mL of TNF-α. In some embodiments, the TIL composition produces 250 pg / mL of TNF-α. In some embodiments, the TIL composition produces 300 pg / mL of TNF-α. In some embodiments, the TIL composition produces 350 pg / mL of TNF-α. In some embodiments, the TIL composition produces 400 pg / mL of TNF-α. In some embodiments, the TIL composition produces 450 pg / mL of TNF-α. In some embodiments, the TIL composition produces 500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 600 pg / mL of TNF-α. In some embodiments, the TIL composition produces 700 pg / mL of TNF-α. In some embodiments, the TIL composition produces 800 pg / mL of TNF-α. In some embodiments, the TIL composition produces 900 pg / mL of TNF-α. In some embodiments, the TIL composition produces 1000 pg / mL of TNF-α. In some embodiments, the TIL composition produces 1500 pg / mL of TNF-α.In some embodiments, the TIL composition produces 2000 pg / mL of TNF-α, hi some embodiments, the TIL composition produces 2500 pg / mL of TNF-α.

[0120] In some embodiments, the TIL composition produces TNF-α after an in vitro autologous tumor assay. In some embodiments, the TIL composition produces 50-100 pg / mL, 50-150 pg / mL, 50-200 pg / mL, 50-400 pg / mL, 50-500 pg / mL, 50-600 pg / mL, 50-700 pg / mL, 50-800 pg / mL, 50-900 pg / mL, or 50-1000 pg / mL of TNF-α. In some embodiments, the TIL composition produces 250-2500 pg / mL, 250-2000 pg / mL, 250-1500 pg / mL, 250-1000 pg / mL, or 250-500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 250 pg / mL, 500 pg / mL, 1000 pg / mL, 1500 pg / mL, 2000 pg / mL, or 2500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 50 pg / mL of TNF-α. In some embodiments, the TIL composition produces 100 pg / mL of TNF-α. In some embodiments, the TIL composition produces 200 pg / mL of TNF-α. In some embodiments, the TIL composition produces 250 pg / mL of TNF-α. In some embodiments, the TIL composition produces 300 pg / mL of TNF-α. In some embodiments, the TIL composition produces 350 pg / mL of TNF-α. In some embodiments, the TIL composition produces 400 pg / mL of TNF-α. In some embodiments, the TIL composition produces 450 pg / mL of TNF-α. In some embodiments, the TIL composition produces 500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 600 pg / mL of TNF-α. In some embodiments, the TIL composition produces 700 pg / mL of TNF-α. In some embodiments, the TIL composition produces 800 pg / mL of TNF-α. In some embodiments, the TIL composition produces 900 pg / mL of TNF-α. In some embodiments, the TIL composition produces 1000 pg / mL of TNF-α. In some embodiments, the TIL composition produces 1500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 2000 pg / mL of TNF-α.In some embodiments, the TIL composition produces 2500 pg / mL of TNF-α.

[0121] In some embodiments, cytotoxic activity can be determined based on the ability to produce or secrete granzyme B in a neoantigen reactivity assay, such as an in vitro coculture assay or an in vitro autologous tumor assay. In some embodiments, the TIL composition produces about 10-fold more granzyme B in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present a neoantigen peptide, compared to a bulk TIL composition. In some embodiments, the TIL composition produces about 20-fold more granzyme B, about 30-fold more granzyme B, about 40-fold more granzyme B, or about 50-fold more granzyme B in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present a neoantigen peptide, compared to a bulk TIL composition. In some embodiments, the TIL composition produces about 100-fold more granzyme B compared to the bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 200-fold more granzyme B, about 300-fold more granzyme B, about 400-fold more granzyme B, or about 500-fold more granzyme B compared to the bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 1000-fold more granzyme B compared to the bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, the TIL composition produces about 2000 times more granzyme B, about 3000 times more granzyme B, about 4000 times more granzyme B, or about 5000 times more granzyme B compared to bulk TIL composition in an in vitro coculture assay, for example, after culture with autologous APCs (e.g., DCs or B cells) that present neo-antigenic peptides.In some embodiments, the TIL composition produces approximately 10,000 times more granzyme B compared to bulk TIL composition in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) presenting neoantigenic peptides.

[0122] In some embodiments, the TIL composition produces about 10 times more granzyme B in an in vitro autologous tumor assay compared to bulk TIL composition. In some embodiments, the TIL composition produces about 20 times more granzyme B, about 30 times more granzyme B, about 40 times more granzyme B, or about 50 times more granzyme B in an in vitro autologous tumor assay compared to bulk TIL composition. In some embodiments, the TIL composition produces about 100 times more granzyme B in an in vitro autologous tumor assay compared to bulk TIL composition. In some embodiments, the TIL composition produces about 200 times more granzyme B, about 300 times more granzyme B, about 400 times more granzyme B, or about 500 times more granzyme B in an in vitro autologous tumor assay compared to bulk TIL composition. In some embodiments, the TIL composition produces about 1000 times more granzyme B in an in vitro autologous tumor assay compared to bulk TIL composition. In some embodiments, the TIL composition produces about 2000 times more granzyme B, about 3000 times more granzyme B, about 4000 times more granzyme B, or about 5000 times more granzyme B in an in vitro autologous tumor assay compared to bulk TIL composition. In some embodiments, the TIL composition produces about 10,000 times more granzyme B in an in vitro autologous tumor assay compared to bulk TIL composition.

[0123] In some embodiments, the TIL composition produces granzyme B in an in vitro coculture assay, e.g., in culture with autologous APCs (e.g., DCs or B cells) presenting neoantigenic peptides. In some embodiments, the TIL composition produces 5,000-10,000 pg / mL, 5,000-15,000 pg / mL, 5,000-20,000 pg / mL, 5,000-25,000 pg / mL, 5,000-30,000 pg / mL, or 5,000-75,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 50,000-600,000 pg / mL, 50,000-500,000 pg / mL, 50,000-400,000 pg / mL, 50,000-300,00 pg / mL, 200,00-500 pg / mL of granzyme B, or 200,000-500 pg / mL of granzyme B. In some embodiments, the TIL composition produces 5,000 pg / mL, 10,000 pg / mL, 20,000 pg / mL, 25,000 pg / mL, 50,000 pg / mL, 75,000 pg / mL, or 100,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 50,000 pg / mL, 100,000 pg / mL, 200,000 pg / mL, 300,000 pg / mL, 400,000 pg / mL, 500,000 pg / mL, or 600,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 5,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 10,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 20,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 25,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 50,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 55,000 pg / mL of Granzyme B. In some embodiments, the TIL composition produces 75,000 pg / mL of Granzyme B. In some embodiments, the TIL composition produces 100,000 pg / mL of Granzyme B.In some embodiments, the TIL composition produces 200,000 pg / mL of Granzyme B. In some embodiments, the TIL composition produces 300,000 pg / mL of Granzyme B. In some embodiments, the TIL composition produces 400,000 pg / mL of Granzyme B. In some embodiments, the TIL composition produces 500,000 pg / mL of Granzyme B. In some embodiments, the TIL composition produces 600,000 pg / mL of Granzyme B.

[0124] In some embodiments, the TIL composition produces granzyme B in an autologous tumor assay. In some embodiments, the TIL composition produces 200-3,000 pg / mL, 200-1,000 pg / mL, or 200-500 pg / mL of granzyme B. In some embodiments, the TIL composition produces 300-3,000 pg / mL, 300-1,000 pg / mL, or 300-500 pg / mL of granzyme B. In some embodiments, the TIL composition produces 5,000-10,000 pg / mL, 5,000-15,000 pg / mL, 5,000-20,000 pg / mL, 5,000-25,000 pg / mL, 5,000-30,000 pg / mL, or 5,000-75,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 200 pg / mL, 500 pg / mL, 1,000 pg / mL, or 3,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 1,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 3,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 5,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 10,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 20,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 25,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 50,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 55,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 75,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 100,000 pg / mL of granzyme B.

[0125] In some embodiments, the provided TIL compositions exhibit a higher degranulation response than bulk TIL compositions in neoantigen reactivity assays, such as in vitro coculture assays or in vitro autologous tumor assays. In some embodiments, among the CD8+ T cells in the provided TIL compositions, more than or about 15%, more than or about 20%, more than or about 30%, more than or about 40%, or more than or about 50% exhibit degranulation in in vitro coculture assays, for example, after culture with autologous APCs (e.g., DCs or B cells) that present neoantigen peptides. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, among the CD8+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% express CD107a in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, among the CD8+ T cells in a provided TIL composition, greater than or about 10% express CD107a in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, among the CD8+ T cells in a provided TIL composition, greater than or about 20% express CD107a in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, among the CD8+ T cells in the provided TIL compositions, more than or about 25% express CD107a in an in vitro co-culture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present neo-antigenic peptides.

[0126] In some embodiments, among the CD4+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% exhibit degranulation in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigen peptide. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, among the CD4+ T cells in a provided TIL composition, greater than or about 15%, greater than or about 20%, greater than or about 30%, greater than or about 40%, or greater than or about 50% express CD107a in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigen peptide. In some embodiments, among the CD4+ T cells in a provided TIL composition, greater than 5% or greater than about 5% express CD107a in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, among the CD4+ T cells in a provided TIL composition, greater than 10% or greater than about 10% express CD107a in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide. In some embodiments, among the CD8+ T cells in a provided TIL composition, greater than 15% or greater than about 15% express CD107a in an in vitro coculture assay, e.g., after culture with autologous APCs (e.g., DCs or B cells) that present the neo-antigenic peptide.

[0127] In some embodiments, among the CD8+ T cells in the provided TIL compositions, more than or about 15%, more than or about 20%, more than or about 30%, more than or about 40%, or more than or about 50% exhibit degranulation in an in vitro autologous tumor assay. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, among the CD8+ T cells in the provided TIL compositions, more than or about 15%, more than or about 20%, more than or about 30%, more than or about 40%, or more than or about 50% express CD107a in an in vitro autologous tumor assay. In some embodiments, among the CD8+ T cells in the provided TIL compositions, more than or about 10% express CD107a in an in vitro autologous tumor assay. In some embodiments, greater than or about 20% of the CD8+ T cells in a provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, greater than or about 25% of the CD8+ T cells in a provided TIL composition express CD107a in an in vitro autologous tumor assay.

[0128] In some embodiments, more than or about 15% of the CD4+ T cells in a provided TIL composition, more than or about 20%, more than or about 30%, more than or about 40%, or more than or about 50% of the CD4+ T cells in a provided TIL composition, exhibit degranulation in an in vitro autologous tumor assay. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, more than or about 15%, more than or about 20%, more than or about 30%, more than or about 40%, or more than or about 50% of the CD4+ T cells in a provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, more than or about 5% of the CD4+ T cells in a provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, greater than or about 10% of the CD4+ T cells in a provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, greater than or about 15% of the CD8+ T cells in a provided TIL composition express CD107a in an in vitro autologous tumor assay.

[0129] In some embodiments, the TIL composition is characterized by killing tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 30% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 40% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 50% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 60% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 70% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 80% of tumor cells in an in vitro autologous tumor assay.

[0130] In certain embodiments, the number of such cells in the composition is a therapeutically effective amount. The effective amount of cells may vary depending on the patient and the type, severity, and extent of the disease. Therefore, a physician can determine what the effective amount is after considering the subject's health status, the extent and severity of the disease, and other variables. In some embodiments, the amount is an amount that reduces the severity, duration, and / or symptoms associated with cancer in an animal. In some embodiments, a therapeutically effective amount is a dose of cells that reduces the growth or spread of cancer in a patient or animal administered a composition described herein by at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% compared to the growth or spread of cancer in a patient (or animal) or group of patients (or animals) that have not been administered the composition. In some embodiments, a therapeutically effective amount is an amount that produces cytotoxic activity that results in the activity of inhibiting or reducing cancer cell proliferation.

[0131] In some embodiments, the TIL compositions provided herein enriched for tumor-reactive cells are 5 pieces or about 10 5 From 10 12 pieces or about 10 12 In some embodiments, the TIL compositions provided herein enriched for tumor-reactive cells comprise an amount of cells equal to 10 5 pieces or about 10 5 From 10 8 pieces or about 10 8 In some embodiments, the TIL compositions provided herein enriched for tumor-reactive cells comprise 10 6 pieces or about 10 6 From 10 12 pieces or about 10 12 In some embodiments, the TIL compositions provided herein enriched for tumor-reactive cells comprise 10 8pieces or about 10 8 From 10 11 pieces or about 10 11 In some embodiments, the TIL compositions provided herein enriched for tumor-reactive cells comprise 10 9 pieces or about 10 9 From 10 10 pieces or about 10 10 In some embodiments, the TIL compositions provided herein enriched for tumor-reactive cells comprise 10 5 More than 10 5 More than or about 10 5 More than 10 cells 6 More than 10 6 More than or about 10 6 More than 10 cells 7 More than 10 7 More than or about 10 7 More than 10 cells 8 More than 10 8 More than or about 10 8 More than 10 cells 9 More than 10 9 More than or about 10 9 More than 10 cells 10 More than 10 10 More than or about 10 10 More than 10 cells 11 More than 10 11 More than or about 10 11 More than 10 cells, or 10 12 More than 10 12 More than or about 10 12 In some embodiments, such amounts can be administered to a subject having a disease or condition, for example, a cancer patient.

[0132] In some embodiments, the volume of the composition is at least 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, e.g., 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 5 In some embodiments, the composition is at least 1 x 10 5 cells / mL, 5 x 10 5 cells / mL, 1 x 10 6 cells / mL, 5 x 10 6 cells / mL, 1 x 10 7 cells / mL, 5 x 10 7 cells / mL or 1 x 10 8 cells / mL, or at least approximately 1 x 10 5 cells / mL, 5 x 10 5 cells / mL, 1 x 10 6 cells / mL, 5 x 10 6 cells / mL, 1 x 10 7 cells / mL, 5 x 10 7 cells / mL or 1 x 10 8 In some embodiments, the composition has a cell density of 1 x 10 cells / mL. 5 cells / mL ~ 1 x 10 8 cells / mL, 1 x 10 5 cells / mL ~ 1 x 10 7 cells / mL, 1 x 10 5 cells / mL ~ 1 x 10 6 cells / mL, 1 x 10 6 cells / mL ~ 1 x 10 7 cells / mL, 1 x 106 cells / mL ~ 1 x 10 8 cells / mL, 1 x 10 6 cells / mL ~ 1 x 10 7 cells / mL or 1 x 10 7 cells / mL ~ 1 x 10 8 cells / mL, or approximately 1 x 10 5 cells / mL ~ 1 x 10 8 cells / mL, approximately 1 x 10 5 cells / mL ~ 1 x 10 7 cells / mL, approximately 1 x 10 5 cells / mL ~ 1 x 10 6 cells / mL, approximately 1 x 10 6 cells / mL ~ 1 x 10 7 cells / mL, approximately 1 x 10 6 cells / mL ~ 1 x 10 8 cells / mL, approximately 1 x 10 6 cells / mL ~ 1 x 10 7 cells / mL or approximately 1 x 10 7 cells / mL ~ 1 x 10 8 cells / mL (inclusive).

[0133] Among the compositions are pharmaceutical compositions and formulations, for example, for administration for adoptive cell therapy. In some embodiments, the cells are formulated with a pharmaceutically acceptable carrier.

[0134] Pharmaceutically acceptable carriers can include any solvent, dispersion medium, coating, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. Supplementary active compounds can also be incorporated into the composition. Pharmaceutical carriers should be suitable for cells, such as saline, dextrose solution, or solutions containing human serum albumin.

[0135] In some embodiments, a pharmaceutically acceptable carrier or vehicle for such compositions is any non-toxic aqueous solution in which cells can be maintained or remain viable for a sufficient period of time to allow for administration of live cells. For example, a pharmaceutically acceptable carrier or vehicle can be saline or buffered saline. A pharmaceutically acceptable carrier or vehicle can also include various biomaterials that can enhance the efficiency of the cells. Cell vehicles and carriers can be, for example, polysaccharides such as methylcellulose (MCTate, DA Shear, SW Offman, DG Stein, MC LaPlaca, Biomaterials 22, 1113, 2001, each of which is incorporated by reference in its entirety), chitosan (Suh JKF, Matthew HW T. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford DS, et al., J Biomed Mater Res, 51, 586, 2000, each of which is incorporated by reference in its entirety), N-isopropylacrylamide copolymer P(NIPAM-co-AA) (YH Bae, B. Vernon, CK Han, SW Kim, J. Control. Release, 2004, 2005, each of which is incorporated by reference in its entirety), or cellulose acetate (Cellulose, 2004). 53, 249, 1998; H. Gappa, M. Baudys, J. J. Koh, S. W. Kim, Y. H. Bae, Tissue Eng. 7, 35, 2001), as well as poly(oxyethylene) / poly(D,L-lactic-co-glycolic acid) (B. Jeong, K. M. Lee, A. Gutowska, Y. H. An, Biomacromolecules 3, 865, 2002, which is incorporated herein by reference in its entirety), P(PF-co-EG) (Suggs L. J., Mikos A. G. Cell Trans, 8, 345, 1999, which is incorporated herein by reference in its entirety), PEO / PEG (Mann B. K., Gobin A. S., Tsai A. T., Schmedlen R. H., West J. L., Biomaterials, 22, 3045, 2001; Bryant S. J., Anseth K. S., each of which is incorporated herein by reference in its entirety).Biomaterials, 22, 619, 2001), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymers, 61, 348, 2005, which is incorporated herein by reference in its entirety), collagen (Lee CR, Grodzinsky AJ, Spector M., Biomaterials 22, 3145, 2001, which is incorporated herein by reference in its entirety), and alginate (Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney D J. Biotech Prog 17, 945, 2001; Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990, each of which is incorporated herein by reference in its entirety).

[0136] In some embodiments, compositions, including pharmaceutical compositions, are sterile. In some embodiments, cell isolation or enrichment is performed in a closed or sterile environment, for example, to minimize errors, user handling and / or contamination. In some embodiments, sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0137] Also provided herein are compositions suitable for cryopreserving provided T cells, including tumor-reactive T cells. In some embodiments, the compositions include a cryoprotectant. In some embodiments, the cryoprotectant is or includes DMSO and / or glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, e.g., ultra-low temperatures, e.g., at temperatures ranging from -40°C to -150°C, e.g., at or about 80°C ± 6.0°C.

[0138] Also provided herein are frozen compositions containing any of the provided TIL compositions and a cryoprotectant.

[0139] In some embodiments, cryopreserved cells are prepared for administration by thawing.In some cases, cells can be administered to a subject immediately after thawing.In such embodiments, the composition can be used immediately without further processing.In other cases, after thawing, cells are further processed, for example, by resuspension with a pharmaceutically acceptable carrier, incubation with an activator or stimulatory agent, or activated, washed, and resuspended in a pharmaceutically acceptable buffer before administration to a subject.

[0140] II. Ex vivo methods for generating TIL compositions enriched for tumor-reactive T cells Various embodiments of the methods provided involve the ex vivo expansion and production of T cell therapy compositions, particularly for use in connection with the treatment of cancer. In some embodiments, the production methods involve the growth and manipulation of patient cells outside the body.

[0141] In some embodiments, provided methods for generating a TIL composition, such as any described herein, include providing tumor cells dissociated from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first T cell population comprising CD4+ T cells and CD8+ T cells; culturing the first T cell population with recombinant IL-2 added at a concentration of 3000 IU / mL to 6000 IU / mL, inclusive, for 14 to 28 days to generate a second T cell population; and co-culturing the second T cell population with autologous antigen-presenting cells (APCs) for 12 to 48 hours with recombinant IL-2 added at a concentration of 100 IU / mL to 1000 IU / mL, wherein the APCs are The method includes the steps of: carrying a pool of tumor-derived peptide neoantigens, each peptide being 13 to 40 amino acids in length, at a concentration of 100 ng / mL per peptide, and providing a ratio of the second T cell population to APCs of 2:1 to 10:1; selecting cells that are surface-positive for CD134 and / or CD137 from the third T cell population to generate a fourth T cell population; and expanding tumor-infiltrating lymphocytes (TILs) by incubating irradiated human peripheral blood mononuclear cells (iPBMCs) with the fourth T cell population at a ratio of 100:500 iPBMCs to cells of the fourth T cell population for 12 to 16 days with recombinant IL-2 added at a concentration of 3000 IU / mL to 6000 IU / mL (inclusive) and 10 to 50 ng / mL of anti-CD3 antibody (OKT3), to produce a therapeutic composition of TILs enriched for tumor-reactive cells.

[0142] In some embodiments, a method for generating a TIL composition can include ex vivo co-culture, incubating a second T cell population with APCs, e.g., autologous APCs, that have been exposed to or contacted with one or more peptides, e.g., synthetic peptides, under conditions in which the APCs are induced to present one or more peptides from tumor-associated antigens. In some embodiments, the T cell population is autologous T cells obtained from a tumor-bearing subject, and the source of the synthetic peptides is a tumor antigen peptide derived from the tumor antigen of the same subject. In some embodiments, the cells obtained from the ex vivo co-culture are a cell population (third population) containing 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.

[0143] In some cases, tumor-reactive T cells can be further enriched by separating or selecting cells that express one of CD137, CD134, or CD137 and CD134 (also referred to as "CD134 and / or CD137").

[0144] In certain embodiments, enriched or isolated T cells from the co-culture, such as after separation or selection of tumor-reactive T cells for cells that express or are surface-positive for CD134 and / or CD137, undergo a second expansion (also referred to as a "rapid expansion protocol" or REP). The second expansion includes incubation with an anti-CD3 antibody (e.g., OKT3), irradiated peripheral blood mononuclear cells (iPBMCs), and recombinant IL-2 to further stimulate the T cells for expansion. The T cells are expanded for a number of days, as desired, and / or until the therapeutic or harvest dose is met. A composition of the expanded T cells can then be harvested and formulated for administration to a subject for treatment of the subject's cancer.

[0145] In certain embodiments, the incubation or culture of T cells is also carried out using a nutrient-containing medium so that the cells can survive outside the body. In embodiments of the provided methods, one or more of the steps can be carried out in a serum-containing medium, such as a medium containing human AB serum. In embodiments of the provided methods, one or more of the steps can be carried out in a 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%, 2%, 3%, 4%, or 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.

[0146] A. Neoantigen Identification and Peptide Generation The provided methods involve in silico generating or identifying a plurality of peptides (also referred to as "P" or "n-mers") comprising at least one cancer-specific cancer neo-antigen. In some embodiments, at least one synthetic peptide is prepared using sequence information from all or a subset of the neo-antigen sequences, and the synthetic peptide is then used in a method for enriching tumor-reactive T cells according to the provided methods.

[0147] In some embodiments, cancer-specific cancer neoepitopes are determined by identifying or isolating tumor-associated antigens or peptide sequences thereof from cancer cells obtained from a subject. The cancer cells can be obtained from any body sample derived from a patient that contains or is expected to contain tumor or cancer cells. The body sample can be any tissue sample, such as blood, tissue sample obtained from a primary tumor or tumor metastasis, lymph node sample, or any other sample containing tumor or cancer cells.

[0148] In some embodiments, the tumor is a hematological tumor.Non-limiting examples of hematological tumors include leukemia, for example, acute leukemia (such as l lq23 positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0149] In some embodiments, the tumor is a solid tumor. Non-limiting examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma, and lobular carcinoma of the breast), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, and thyroid carcinoma. carcinoma), papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some examples, the tumor is melanoma, lung cancer, lymphoma, breast cancer, or colon cancer.

[0150] In some embodiments, the tumor is derived from a patient with cancer, including but not limited to ovarian, vulvar, endometrial, urothelial, breast, colorectal, lung, renal, and skin (including but not limited to melanoma). In some embodiments, the tumor is derived from a patient with ovarian cancer. In some embodiments, the tumor is derived from a patient with vulvar cancer. In some embodiments, the tumor is derived from a patient with endometrial cancer. In some embodiments, the tumor is derived from a patient with urothelial cancer. In some embodiments, the tumor is derived from a patient with breast cancer. In some embodiments, the tumor is derived from a patient with colorectal cancer. In some embodiments, the tumor is derived from a patient with lung cancer. In some embodiments, the tumor is derived from a patient with renal cancer. In some embodiments, the tumor is derived from a patient with melanoma. In certain embodiments, the tumor is derived from a patient treated as described in Section III.

[0151] In some embodiments, the cancer is a cancer of the gastrointestinal (GI tract), e.g., cancer or GI cancer involving the upper or lower GI tract, or accessory organs of digestion, such as the esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum, or anus. In some embodiments, the cancer is esophageal cancer, stomach (gastric) cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, cancer of the mucosa-associated lymphoid tissue (MALT lymphoma), cancer of the bile duct, colorectal cancer (including colon cancer, rectal cancer, or both), anal cancer, or a GI carcinoid tumor.

[0152] In a particular embodiment, the cancer is colorectal cancer.

[0153] In some embodiments, the tumor originates from a breast cancer, such as ductal carcinoma or lobular carcinoma. In some embodiments, the tumor originates from prostate cancer. In some embodiments, the tumor originates from a skin cancer, such as basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, or melanoma. In some embodiments, the tumor originates from a lung cancer, such as adenocarcinoma, bronchioloalveolar carcinoma, large cell carcinoma, or small cell carcinoma. In some embodiments, the tumor originates from a brain cancer, such as glioblastoma or meningioma. In some embodiments, the tumor originates from a gastrointestinal cancer, such as any of the above. In some embodiments, the tumor originates from colon cancer. In some embodiments, the tumor originates from a liver cancer, such as hepatocellular carcinoma. In some embodiments, the tumor originates from pancreatic cancer. In some embodiments, the tumor originates from a kidney cancer, such as renal cell carcinoma. In some embodiments, the tumor originates from testicular cancer.

[0154] In some embodiments, the cancer is not melanoma. Melanoma is a cancer that generally has a high mutation rate. A high tumor mutation burden has been considered a particularly desirable prognostic marker for the success of immunotherapy treatment targeting tumor neoantigens (Simpson et al., Journal of Clinical Oncology 2017, 35:15_suppl, 9567-9567; McGranahan et al. Science 2016, 351:1463-1469). In some embodiments, the provided methods are performed to actively (rather than passively) enrich tumor-reactive T cells, and therefore can be used for cancers with a relatively low tumor mutation burden.

[0155] In some embodiments, the subject has a tumor mutational burden of 5-6000 mutations. In some embodiments, the subject has a tumor mutational burden of 100-5500 mutations. In some embodiments, the subject has a tumor mutational burden of about 100 mutations, about 200 mutations, about 300 mutations, about 400 mutations, about 500 mutations, about 600 mutations, about 700 mutations, about 800 mutations, about 900 mutations, about 1000 mutations, about 1500 mutations, about 2000 mutations, about 2500 mutations, about 3000 mutations, about 3500 mutations, about 4000 mutations, about 4500 mutations, about 5000 mutations, about 5500 mutations, or any value in between any of the foregoing.

[0156] In some embodiments, the subject has a tumor mutational burden (TMB) of less than 8 mutations.

[0157] In some embodiments, TMB includes the number of nonsynonymous mutations per tumor. In some embodiments, TMB can be calculated by counting the number of synonymous and nonsynonymous mutations over a 0.8-1.2 megabase (Mb) region and reporting the results as mutations / Mb. In some embodiments, TMB can be determined by next-generation sequencing (NGS) on tumor tissue samples. In some cases, whole-exome sequencing can be used, or computational germline status filtering can be used (Chalmers et al. Genome Med 2017 9:34). In some embodiments, the subject has a TMB of less than or about 60 mutations / Mb, e.g., less than or about 55 mutations / Mb, less than or about 55 mutations / Mb, less than or about 50 mutations / Mb, less than or about 45 mutations / Mb, less than or about 40 mutations / Mb, less than or about 30 mutations / Mb, less than or about 25 mutations / Mb, or less than or about 20 mutations / Mb, or any value between any of the foregoing. In some embodiments, the subject has a TMB of less than or about 41 mutations / Mb, less than or about 41 mutations / Mb, less than or about 40 mutations / Mb, less than or about 39 mutations / Mb, less than or about 38 mutations / Mb, less than or about 37 mutations / Mb, or less.

[0158] In some embodiments, the peptide (P) is a tumor-associated antigen derived from a variant of carcinoma in situ or a pre-malignant condition such as vulvar intraepithelial neoplasia, cervical intraepithelial neoplasia, or vaginal intraepithelial neoplasia.

[0159] In some aspects, the nucleic acid from such tumor or cancer cell is obtained and sequenced.In some embodiments, the protein coding region of gene in genome is obtained by, for example, omics analysis, for example, by analyzing whole genome sequencing data, exome sequencing data and / or transcriptome data.To identify tumor-specific sequence, sequencing data can be compared with reference sequencing data, for example, the data obtained from normal cell or non-cancerous cell from the same subject.In some embodiments, next-generation sequencing (NGS) method is used.

[0160] In some embodiments, the method comprises using matched normal omics data of tumor. In such methods, the in silico analysis comprises omics analysis to identify mutations in tumor compared with normal tissue of the same patient, for example, non-disease tissue of the same patient. The matched normal omics data is whole genome sequencing data, exome sequencing data and / or transcriptome data, and the matched normal omics data is generally considered to be matched with normal before the patient's treatment. In certain embodiments, whole exome sequencing is performed on healthy tissue and diseased tissue to identify somatic mutations associated with tumor.

[0161] In some embodiments, the omics data is obtained from one or more patient biopsy samples according to standard tissue processing and sequencing protocols. In certain embodiments, the data is patient-matched tumor data (e.g., tumor vs. normal from the same patient). In some cases, unmatched or matched data to other references (e.g., previous normals from the same patient, or previous tumors from the same patient, or homostatistics) are also considered suitable for use herein. The omics data can be new omics data or omics data obtained from a previous procedure (or a different patient). For example, neoepitopes can be identified in a first step from a patient tumor by whole genome and / or exome analysis of a tumor biopsy (or lymphoid biopsy, or biopsy from a metastatic site) and matched normal tissue (i.e., non-diseased tissue obtained from the same patient, such as peripheral blood). In some embodiments, genomic analysis can be processed through position-guided synchronous comparison of the omics information thus obtained.

[0162] Genome analysis can be performed by any number of analytical methods. In certain embodiments, the method includes WGS (whole genome sequencing) and exome sequencing of both tumor and matched normal samples using next-generation sequencing such as massively parallel sequencing, ion torrent sequencing, and pyrosequencing. Computational analysis of sequence data can be performed in many ways. In some embodiments, the data format is SAM, BAM, GAR, or VCF format. As an example, analysis can be performed in silico by position-guided synchronous alignment of tumor and normal samples, for example, as disclosed in US Patent No. 2012 / 0059670 and US Patent No. 2012 / 0066001, using BAM files and a BAM server. Alternative file formats for sequence analysis (e.g., SAM, GAR, FASTA, etc.) are also contemplated.

[0163] In some optional embodiments, peptides (P) containing neoantigens resulting from missense mutations include amino acid changes encoded by one or more nucleotide polymorphisms. Peptides (P) containing neoantigens resulting from frameshift mutations, splice site variants, insertions, inversions, and deletions should include novel peptide sequences and junctions of novel peptide sequences. Peptides (P) containing neoantigens with novel post-translational modifications should include amino acids with post-translational modifications such as phosphates or glycans.

[0164] Once these mutations are identified, neoepitopes are then identified. Neoepitopes are mutant peptides recognized by the patient's T cells. These neoepitopes must be presented by tumors or antigen-presenting cells via the MHC complex and then recognized by TCRs on T cells. In some embodiments, the provided methods include calculating one or more neoepitopes to define tumor- and patient-specific neoepitopes. Thus, it should be appreciated that patient- and cancer-specific neoepitopes can be identified exclusively in silico from omics information that ultimately predicts potential epitopes specific to a patient and tumor type. In certain aspects, such identified cancer neoepitopes are unique to the patient and the patient's particular cancer (e.g., having a frequency of less than 0.1% of all neoepitopes, and more typically less than 0.01% within a population of cancer patients diagnosed with the same cancer), but such identified cancer neoepitopes are likely to be presented in tumors.

[0165] In some optional embodiments, the length of the peptide (P) depends on the specific application and is typically about 5 to about 50 amino acids. In certain embodiments, the neoepitope is calculated to have a length of 2 to 50 amino acids, e.g., 13 to 40 amino acids, e.g., 25 amino acids (25-mer). In preferred embodiments, the peptide (P) is about 13 to 40 amino acids, e.g., 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids.

[0166] In some embodiments, the method is performed using a peptide pool. The peptide pool can contain tens to hundreds of individual peptides. The peptide pool can contain up to 200 different peptides containing predicted mutations. In some cases, the peptide pool contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or more individual peptides, or any value between any of the foregoing.

[0167] A peptide pool can represent one neo-antigen or several neo-antigens. In some cases, a peptide pool can contain multiple overlapping peptides of the same neo-antigen. Thus, in the case of tumor-associated antigens, the antigen can be divided into peptides (P) of 13 to 40 amino acids, e.g., 25 amino acids, where each peptide (P) contains a unique amino acid composition, or the peptides (P) can be overlapping peptide pools in which the antigen is divided into a set number of peptides (P) of 13 to 40 amino acids, e.g., 25 amino acids, with overlapping sequences. In some cases, each peptide in an overlapping pool of antigens can be offset by a set number of amino acid residues, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, or 15 amino acids. In some embodiments, each peptide in an overlapping pool of antigens is offset by 10 amino acids. In some embodiments, each peptide in an overlapping pool of antigens is offset by 12 amino acids. For example, an overlapping peptide pool comprising a 100 amino acid antigen can be divided into eight 25 amino acid peptides (P), each offset by 12 amino acids (i.e., each subsequent 25 amino acid peptide comprising the 100 amino acid peptide sequence begins 13 amino acid positions from the previous peptide). Those skilled in the art will appreciate that there are many permutations for generating peptide pools from an antigen.

[0168] Various algorithms have been developed and can be used to map T cell epitopes (both MHC class I-restricted and MHC class II-restricted) within protein molecules of various origins. In some embodiments, many programs utilize the availability of large-scale peptide-MHC binding affinity matrices from experimental measurements to train machine learning (ML)-based classifiers to distinguish MHC-binders from non-binders (see, e.g., Zhao et al. (2018) PLoS Comput Biol 14(11):e1006457). Exemplary prediction methods for MHC class I (e.g., 9-mer) include smm, smmpmbec, ann(NetMHC3.4), NetMHC4, PickPocket, consensus, NetMHCpan2.8, NetMHCpan3, NetMHCpan4, NetMHCcons, mhcflurry, mhcflurry_pan, or MixMHCpred. Exemplary prediction methods for MHC class II (e.g., 15-mer) include NetMHCIIpan, NetMHCII2.3, nn_align, smm_align, consensus, comblib, tepitope, or mhcflurry. Any of these methods can be used.

[0169] After in silico identification of suitable neoepitope sequences, corresponding synthetic peptides are prepared in vitro (e.g., using solid-phase synthesis). In certain embodiments, a library of synthetic peptides is prepared, representing multiple different neoepitopes from a subject.

[0170] Synthetic peptides can be prepared using a variety of methods. For example, peptides containing cancer neoepitope sequences can be prepared using solid phase (e.g., using Merrified synthesis), via solution phase synthesis, or from relatively small peptide fragments. Peptide epitopes can be obtained by chemical synthesis using commercially available automated peptide synthesizers. In some embodiments, peptides can be synthesized using the Fmoc-polyamide mode of solid phase peptide synthesis, as disclosed, for example, by Lu et al. (1981). J. Org. Chem. 46, 3433 and references therein. In some aspects, peptides can be produced by expression of recombinant nucleic acids using a suitable expression system in a suitable host. In some aspects, recombinant methods can be used in which multiple neoepitopes are present on a single peptide chain, for example, with spacers between neoepitopes or cleavage sites.

[0171] Peptides can be purified by any one or a combination of techniques, such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and reversed-phase high performance liquid chromatography using, for example, acetonitrile / water gradient separation. In some embodiments, peptides can be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Peptide analysis can be performed using thin-layer chromatography, electrophoresis, particularly capillary electrophoresis, solid-phase extraction (CSPE), reversed-phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, fast atom bombardment (FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.

[0172] B. Tumor Samples for First Enrichment The provided methods include obtaining and enriching or selecting a T cell population from a biological sample for use as a first T cell population (also referred to as an input population). In some cases, the first T cell population is known to contain tumor antigen-reactive T cells, or is likely to contain tumor antigen-reactive T cells, or is capable of responding to a tumor antigen, for example, after ex vivo co-culture with an autologous source of tumor antigen. For example, typically, the first T cell population is derived from a biological sample obtained from a tumor or from a subject known to have or likely to have a tumor. In certain embodiments, the first T cell population is further stimulated with one or more T cell stimulators (e.g., one or more recombinant cytokines, such as IL-2) to generate a second T cell population or stimulated T cell population containing expanded T cells after stimulation.

[0173] 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 react to tumor antigens.

[0174] The patient tumor sample 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, the tumor sample is obtained by surgical resection. In some embodiments, the tumor sample is obtained by needle biopsy. Generally, the tumor sample can be derived from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. The tumor sample can also be derived from a liquid tumor, such as a tumor derived from a hematological malignancy. Typically, the tumor sample is derived from the same patient as the tumor sample used for neoantigen identification described above. In some embodiments, the tumor sample is the same tumor sample used for neoantigen identification described above.

[0175] In some embodiments, the tumor sample is derived from a solid tumor, which can be of any cancer type (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma), including, but not limited to, ovarian, vulvar, endometrial, urothelial, breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach (gastrointestinal), and skin. In some embodiments, the tumor is derived from a patient with cancer, including, but not limited to, ovarian, vulvar, endometrial, urothelial, breast, colorectal, lung, kidney, and skin (including, but not limited to, melanoma). In some embodiments, the tumor is derived from a patient with ovarian cancer. In some embodiments, the tumor is derived from a patient with vulvar cancer. In some embodiments, the tumor is derived from a patient with endometrial cancer. In some embodiments, the tumor is derived from a patient with urothelial cancer. In some embodiments, the tumor is derived from a patient with breast cancer. In some embodiments, the tumor is derived from a patient with colorectal cancer. In some embodiments, the tumor is derived from a patient with lung cancer. In some embodiments, the tumor is derived from a patient with renal cancer. In some embodiments, the tumor is derived from a patient with melanoma. In certain embodiments, the tumor is derived from a patient treated as described in Section III.

[0176] In certain embodiments, the T cell population comprises both CD4+ T cells and CD8+ T cells. Many cancers, including solid tumors, including many common epithelial indications (e.g., GI), express class I and class II restricted mutations. For T cell products to target such indications, such as common epithelial indications, it is believed that both CD8+ T cells recognize class I MHC restricted molecules and CD4+ T cells recognize class II MHC restricted molecules.

[0177] 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.

[0178] 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.

[0179] In provided embodiments, the tumor sample obtained is less than 1 mm in size. 3 or about 1 mm 3 From, 8mm 3 or about 8 mm 3 , e.g., 1 mm 3 or about 1 mm 3 From, 3mm 3 or about 3 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 -3 mm 3 In some embodiments, the tumor fragment is about 1 to 3 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.

[0180] In some of any of the provided embodiments, the obtained tumor sample is fragmented into pieces having a diameter of 1 mm or about 1 mm to 8 mm or about 8 mm, e.g., 1 mm or about 1 mm to 6 mm or about 6 mm, 1 mm or about 1 mm to 4 mm or about 4 mm, 1 mm or about 1 mm to 2 mm or about 2 mm. In some embodiments, the tumor fragments are about 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.

[0181] In some embodiments, the tumor sample is cryopreserved prior to fragmentation. In some embodiments, the tumor fragments are cryopreserved.

[0182] In some embodiments, tumor fragments are used as a source for preparing a single cell suspension for use as an input population of T cells in the first expansion in the methods provided.

[0183] In some embodiments, the provided methods include obtaining cells from tumor fragments, for example, by enzymatic digestion of the tumor fragments to obtain TILs. The enzymatic digestion can be performed using a collagenase, such as type IV collagenase or type I / II collagenase. The enzyme, such as collagenase, can be present in the medium for enzymatic digestion at a concentration of 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL, for example, 1 mg / mL or about 1 mg / mL, 2 mg / mL or about 2 mg / mL, 3 mg / mL or about 3 mg / mL, 4 mg / mL or about 4 mg / mL, or 5 mg / mL or about 5 mg / mL, or any value between any of the foregoing. The enzyme, such as collagenase, can be present in the medium for enzymatic digestion at a concentration of from at or about 5 mg / mL to at or about 10 mg / mL, e.g., 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL, or any value between any of the foregoing. In some embodiments, the concentration is about 5 mg / mL. In some embodiments, the concentration is 10 mg / mL. In some embodiments, the collagenase is type IV collagenase. In some embodiments, the collagenase is type I / II collagenase. In some embodiments, the enzymatic digestion is with a medium containing, for example, from at or about 1 mg / mL to at or about 5 mg / mL of type IV collagenase. In some embodiments, the enzymatic digestion is with a medium containing, for example, from 1 mg / mL or about 1 mg / mL to 5 mg / mL or about 5 mg / mL of type I / II collagenase.

[0184] In some embodiments, enzymatic digestion can be carried out in part using hyaluronidase. Hyaluronidase is a hyaluronic acid metabolic enzyme that subsequently enhances cell membrane permeability (Eikenes et al., Anticancer Research, 2010). Enzymes such as hyaluronidase can be present in the medium for enzymatic digestion at a concentration of 5 mg / mL or about 5 mg / mL to 10 mg / mL or about 10 mg / mL, for example, 5 mg / mL or about 5 mg / mL, 6 mg / mL or about 6 mg / mL, 7 mg / mL or about 7 mg / mL, 8 mg / mL or about 8 mg / mL, or 9 mg / mL or about 9 mg / mL, 10 mg / mL or about 10 mg / mL, or any value between the above. In some embodiments, enzymatic digestion is carried out in a medium containing, for example, 5 mg / mL or about 5 mg / mL to 10 mg / mL or about 10 mg / mL of type II hyaluronidase. In some embodiments, if more gentle digestion is desired, 5 mg / mL or about 5 mg / mL of hyaluronidase is used. In some embodiments, if more thorough digestion is desired, a higher concentration of hyaluronidase is used, for example, 10 mg / mL or about 10 mg / mL of hyaluronidase.

[0185] In some embodiments, dNase is also present in the medium during enzymatic digestion. dNase is an enzyme that degrades any free DNA released into the medium as a result of the tumor fragment digestion process. Enzymes such as dNase I can be present in the medium for enzymatic digestion at a concentration of 5,000 units / mL or about 5,000 units / mL to 10,000 units / mL or about 10,000 units / mL, for example, 5,000 units / mL or about 5,000 units / mL, 6,000 units / mL or about 6,000 units / mL, 7,000 units / mL or about 7,000 units / mL, 8,000 units / mL or about 8,000 units / mL, or 9,000 units / mL or about 9,000 units / mL, 10,000 units / mL or about 10,000 units / mL, or any value between the aforementioned. In some embodiments, the enzymatic digestion is with a medium containing, for example, from at or about 5,000 units / mL to at or about 10,000 units / mL of dNase I.

[0186] In other embodiments, enzymes from the Miltenyi Human Tumor Dissociation Kit can be used (e.g., catalog O.130-095-929; Miltenyi Biotec). The enzyme medium containing the enzyme can be a serum-free medium, such as any of those described. In certain embodiments, the enzyme medium contains collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g., GlutaMAX), 10 mg / mL gentamicin, 30 units / mL dNase, and 1.0 mg / mL collagenase. In some embodiments, the enzyme medium contains 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 certain embodiments, the enzyme medium comprises hyaluronidase and / or collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamate (e.g., GlutaMAX), 10 mg / mL gentamicin, 10,000 units / mL dNase I, 10 mg / mL collagenase, and 10 mg / mL hyaluronidase.

[0187] 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 generated by placing the tumor in enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation at 37°C in 5% CO2 for 30 minutes, followed by repeated cycles of mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments are present. In some embodiments, the tumor digest is subjected to homogenization and enzymatic digestion by incubation in an enzyme cocktail for 15 minutes to 2 hours, e.g., 30 minutes to 60 minutes, or about 30 minutes to 60 minutes. In some embodiments, the tumor digest is subjected to homogenization and enzymatic digestion by incubation in an enzyme cocktail for approximately 60 minutes. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, density gradient separation using FICOLL can be performed to remove these cells. In some embodiments, single cell suspension is prepared after processing tumor fragments by straining cells through a filter to remove debris, for example, a 70 μm strainer. In some cases, separation can be achieved by centrifugation, in which case the cell pellet can be resuspended and strained through, for example, 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, the disclosure of which is incorporated herein by reference. Any of the aforementioned methods can be used in any of the embodiments described herein for obtaining TILs for use in the provided methods.

[0188] In some embodiments, a single cell suspension for use as an input population comprises 1 x 10 6 pieces or approximately 1 x 10 6 From 1000 x 10 dissociated tumor cells 6 pieces or approximately 1000 x 10 6 1 x 10 dissociated tumor cells, e.g., 1 x 10 6 ~500×10 61 x 10 dissociated tumor cells 6 ~100×10 6 1 x 10 dissociated tumor cells 6 ~50×10 6 1 x 10 dissociated tumor cells 6 ~10×10 6 10 x 10 dissociated tumor cells 6 ~1000×10 6 10 x 10 dissociated tumor cells 6 ~100×10 6 10 x 10 dissociated tumor cells 6 ~500×10 6 10 x 10 dissociated tumor cells 6 ~50×10 6 50 x 10 dissociated tumor cells 6 ~1000×10 6 50 x 10 dissociated tumor cells 6 ~500×10 6 50 x 10 dissociated tumor cells 6 ~100×10 6 100 x 10 dissociated tumor cells 6 ~1000×10 6 100 x 10 dissociated tumor cells 6 ~500×10 6 dissociated tumor cells, or 500 x 10 6 ~1000×10 6 In some embodiments, the single cell suspension for use as an input population of T cells comprises 10 x 10 dissociated tumor cells. 6 20 x 10 dissociated tumor cells 6 30 x 10 dissociated tumor cells 6 40 x 10 dissociated tumor cells 6 50 x 10 dissociated tumor cells 6 60 x 10 dissociated tumor cells 6 70 x 10 dissociated tumor cells 6 80 x 10 dissociated tumor cells 6 90 x 10 dissociated tumor cells 6 dissociated tumor cells, or 100 x 10 6dissociated tumor cells, or approximately 10 x 10 6 Dissociated tumor cells, approximately 20 x 10 6 Dissociated tumor cells, approximately 30 x 10 6 Dissociated tumor cells, approximately 40 x 10 6 Dissociated tumor cells, approximately 50 x 10 6 Dissociated tumor cells, approximately 60 x 10 6 Dissociated tumor cells, approximately 70 x 10 6 Dissociated tumor cells, approximately 80 x 10 6 Dissociated tumor cells, approximately 90 x 10 6 dissociated tumor cells, or approximately 100 x 10 6 dissociated tumor cells, or at least 10 x 10 6 Dissociated tumor cells, at least 20 x 10 6 Dissociated tumor cells, at least 30 x 10 6 Dissociated tumor cells, at least 40 x 10 6 Dissociated tumor cells, at least 50 x 10 6 Dissociated tumor cells, at least 60 x 10 6 Dissociated tumor cells, at least 70 x 10 6 Dissociated tumor cells, at least 80 x 10 6 Dissociated tumor cells, at least 90 x 10 6 dissociated tumor cells, or at least 100 x 10 6 dissociated tumor cells, or at least about 10 x 10 6 Dissociated tumor cells, at least approximately 20 x 10 6 Dissociated tumor cells, at least approximately 30 x 10 6 Dissociated tumor cells, at least approximately 40 x 10 6 Dissociated tumor cells, at least approximately 50 x 10 6 Dissociated tumor cells, at least approximately 60 x 10 6 Dissociated tumor cells, at least approximately 70 x 10 6 Dissociated tumor cells, at least approximately 80 x 10 6 Dissociated tumor cells, at least approximately 90 x 10 6dissociated tumor cells, or at least about 100 x 10 6 In some embodiments, the single cell suspension for use as an input population of T cells comprises 10 x 10 dissociated tumor cells. 6 pieces or approximately 10 x 10 6 From 100 x 10 dissociated tumor cells 6 pieces or approximately 100 x 10 6 Contains dissociated tumor cells.

[0189] In some embodiments, digested cells from tumor fragments are placed in culture medium with appropriate nutrients for mediating T cell activation and / or maintaining T cell expansion under conditions for mediating T cell activation and / or maintaining T cell expansion, such as any of the conditions described below, for T cell stimulation and pre-expansion. In some embodiments, T cell stimulation involves incubating the first T cell population (e.g., dissociated tumor cells) generated or obtained above 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. Cells are seeded at a specific density appropriate for the specific 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. For a G-Rex 6M (single well) or 10M vessel, 1 x 10 cells are seeded. 7 ~4×10 7 Ideally, a total of 100 cells are seeded. A particular culture vessel can be selected based on the number of cells available and / or the desired yield of cells. The choice of culture vessel (e.g., G-Rex) can be 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 approximately 100 cm. 2(e.g., G-Rex 100 M / 100M-CS) 1. In some embodiments, the surface area of ​​the culture vessel is about 500 cm 2 (For example, G-Rex 500 M / 500M-CS).

[0190] In some of any of the provided embodiments, the biological sample is a tumor-derived sample, and the number of cells at the start of the culturing step is 10 x 10 6 pieces or approximately 10 x 10 6 From 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 total viable cells, or any value between any of the aforementioned.

[0191] In some embodiments, IL-2 is added to the culture medium for expansion. In some embodiments, the culture medium is a complete medium. In some embodiments, the culture medium is a serum-free medium. In some embodiments, the culture medium is a serum-free medium containing recombinant IL-2.

[0192] In some embodiments, recombinant IL-2 is present in the cell culture medium. IL-2 is a cytokine that supports the recovery and proliferation of T cells. IL-2 also supports T cell homeostasis, thereby supporting their phenotype, differentiation state, and immunological memory. In some cases, the induction of 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).

[0193] In some embodiments, recombinant IL-2 is administered at a concentration of from 1000 IU / mL or about 1000 IU / mL, 8000 IU / mL or about 8000 IU / mL, e.g., from 1000 IU / mL or about 1000 IU / mL, 7000 IU / mL or about 7000 IU / mL, 1000 IU / mL or about 1000 IU / mL, 6000 IU / mL or about 6000 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, 2000 IU / mL or about 2000 IU / mL, 8000 IU / mL or about 8000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 7000 IU / mL or about 7000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 6000 IU / mL or about 6000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 5000 IU / mL or is from about 5000 IU / mL, 2000 IU / mL or about 2000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 8000 IU / mL or about 8000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 7000 IU / mL or about 7000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 6000 IU / mL or about 6000 IU / mL, 4000 IU / mL or about 4000 IU / mL, 5000 IU / mL or about 5000 IU / mL, from 000 IU / mL or about 5000 IU / mL, 8000 IU / mL or about 8000 IU / mL, 5000 IU / mL or about 5000 IU / mL, 7000 IU / mL or about 7000 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, 8000 IU / mL or about 8000 IU / mL, 6000 IU / mL or about 6000 IU / mL, 7000 IU / mL or about 7000 IU / mL,Alternatively, recombinant IL-2 is added to the culture medium at a concentration of at or about 7000 IU / mL to at or about 8000 IU / mL. In some embodiments, recombinant IL-2 is present in an amount of at or about 6000 IU / mL. In some embodiments, recombinant IL-2 is present in an amount of at or about 3000 IU / mL.

[0194] In some embodiments, incubation with a T cell stimulator is carried out under conditions for the initial expansion of T cells from the biological sample. In some embodiments, the cells are cultured at about 37° C. with about 5% CO2.

[0195] In some embodiments, incubation with the T cell stimulatory agent is for 7 to 28 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, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, or any value between any of the foregoing. In some embodiments, incubation is for 7 to 28 days. In some embodiments, incubation is for 7 to 14 days.

[0196] In some embodiments, the first T cell population is cultured for 14-28 days with recombinant IL-2 added at a concentration of 3000 IU / mL to 6000 IU / mL (inclusive) to generate a second T cell population. In some embodiments, the first T cell population is cultured for 14-28 days with recombinant IL-2 added at a concentration of about 3000 IU / mL to generate a second T cell population. In some embodiments, the first T cell population is cultured for 14-28 days with recombinant IL-2 added at a concentration of about 6000 IU / mL to generate a second T cell population. In some embodiments, the first T cell population is cultured for about 14 days. In some embodiments, the first T cell population is cultured for about 15 days. In some embodiments, the first T cell population is cultured for about 16 days. In some embodiments, the first T cell population is cultured for about 17 days. In some embodiments, the first T cell population is cultured for about 18 days. In some embodiments, the first T cell population is cultured for about 19 days. In some embodiments, the first T cell population is cultured for about 20 days. In some embodiments, the first T cell population is cultured for about 21 days. In some embodiments, the first T cell population is cultured for about 22 days. In some embodiments, the first T cell population is cultured for about 23 days. In some embodiments, the first T cell population is cultured for about 24 days. In some embodiments, the first T cell population is cultured for about 25 days. In some embodiments, the first T cell population is cultured for about 26 days. In some embodiments, the first T cell population is cultured for about 27 days. In some embodiments, the first T cell population is cultured for about 28 days.

[0197] In some cases, the medium can be changed daily, every other day, every third day, every fourth day, or once a week during the culture or incubation period. In some embodiments, recombinant IL-2 is supplemented (added) at each medium change. In some embodiments, about 3000 IU / mL of recombinant IL-2 is supplemented (added) at each medium change. In some embodiments, about 6000 IU / mL of recombinant IL-2 is supplemented (added) at each medium change.

[0198] For example, incubation for initial expansion of T cells in a biological 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 a serum-free medium. In some embodiments, the incubation is performed in a closed, automated system using a serum-free medium.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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 IA, and / or APC generation, as described in Section IC. 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.

[0203] 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.

[0204] 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, 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.

[0205] C. Co-culture with APCs In an embodiment of the provided method, once the neoepitope-encoding protein has been synthesized into multiple synthetic peptides, the pool of synthetic peptides is contacted with antigen-presenting cells under conditions that present the peptides in the context of MHC molecules. The antigen-presenting cells are used to present these peptides. The peptide-loaded APCs are then co-cultured with T cells from a second T cell population generated after initial expansion (pre-expansion) of T cells with one or more stimulants. For recognition of the peptides presented on the APCs, the loaded APCs (presenting the peptides) are incubated with T cells from the second population of pre-expanded T cells. T cells that recognize these peptides on the surface of the APCs can then be isolated or selected from the co-culture, for example, by the methods described below. In a specific embodiment, the co-culture of the second T cell population is with autologous antigen-presenting cells (APCs).

[0206] In some embodiments of the provided methods, the methods may include co-culturing T cells with APCs for several hours to several days, followed by separating antigen-presenting cells from the T cell population to expand the T cells under conditions to enrich or expand tumor-reactive T cells. In some embodiments, the separating step may include isolating or selecting reactive T cells from the culture based on one or more T cell activation markers on the T cells, e.g., CD134 and / or CD137.

[0207] Various methods can be used to culture cells for antigen specificity, see, e.g., U.S. Patent Application Publication No. 2017 / 0224800.

[0208] In some embodiments, tumor-reactive T cells are co-cultured with APCs that have been contacted or exposed to present a peptide containing a mutant amino acid sequence, e.g., the neoepitope peptide described above. The method may include inducing the patient's autologous antigen-presenting cells (APCs) to present the mutant amino acid sequence. APCs may include any cells that present peptide fragments of proteins associated with major histocompatibility complex (MHC) molecules on their cell surface. MHC molecules may be any MHC molecule expressed by the patient, including, but not limited to, MHC class I molecules, MHC class II molecules, HLA-A molecules, HLA-B molecules, HLA-C molecules, HLA-DM molecules, HLA-DO molecules, HLA-DP molecules, HLA-DQ molecules, and HLA-DR molecules. APCs may include, for example, any one or more of macrophages, DCs, Langerhans cells, B lymphocytes, and T cells. In certain embodiments, the APCs are DCs. In some specific embodiments, the APCs are B cells. In some embodiments, the APCs are artificial APCs.

[0209] In certain embodiments, APCs comprise cells that can present class I-restricted molecules and class II-restricted molecules. For example, both B cells and DCs have the ability to present MHC class I-restricted molecules and MHC class II-restricted molecules. In some embodiments, APC cell samples comprise B cells and DCs. In some embodiments, APC cell samples are enriched for B cells, for example, by selection or isolation from primary cell samples. In some embodiments, APC cell samples are enriched for DCs, for example, by selection or isolation from primary cell samples.

[0210] In some embodiments, the APC expresses MHC class I molecules and / or MHC class II molecules with the matching HLA from which the T cell source was obtained. In certain embodiments, both the APC and the T cell are isolated from the same subject, i.e., are autologous to the cancer patient. In some embodiments, the method may include inducing the patient's autologous antigen-presenting cells (APCs) to present the mutant amino acid sequence. By using the autologous APCs obtained from the patient, the method can identify T cells with antigen specificity for the mutant amino acid sequence encoded by the cancer-specific mutation, which is presented in the context of the MHC molecules expressed by the patient.

[0211] In some embodiments, the APCs are cells from a blood sample or an apheresis sample from a subject, such as a patient. In some embodiments, the APCs comprise cells present in a peripheral blood mononuclear cell (PBMC) sample. Typically, APCs function in PBMC cultures that primarily contain monocytes and B cells. In some embodiments, a population of isolated PBMCs can be used as APCs in the provided methods. PBMCs can be obtained using standard methods, such as Ficoll-Paque gradient separation. In some cases, the APCs are or comprise B cells isolated from a blood or apheresis sample, or from a PBMC sample. In other cases, the APCs are or comprise monocytes isolated from a blood or apheresis sample, or from a PBMC sample. In some aspects, monocytes can be used as a source for preparing monocyte-derived DCs for use as APCs. In some embodiments, monocyte-derived DCs (e.g., CD11c high MHCII high CD14 low A source of monocyte-derived dendritic cells (DCs) can be generated ex vivo from isolated monocytes by culturing them with GM-CSF and IL-4 for 4-6 days to generate monocyte-derived dendritic cells. In certain embodiments, monocytes are isolated from PBMCs, such as by CD14 selection, and then cultured with GM-CSF and IL-4 for 4-6 days.

[0212] In some embodiments, the APCs are replication-competent primary cells (e.g., B cells or monocyte-derived DCs), e.g., the cells are not subjected to irradiation, heat treatment, or other methods that result in their inactivation. In certain embodiments, the provided methods do not use irradiated APCs. In some embodiments, the APCs are freshly isolated primary cells obtained from the subject, or are derived from primary cells obtained from the subject. In some embodiments, the APCs have been cryopreserved and then thawed prior to co-culture with stimulated T cells according to the provided methods.

[0213] In some embodiments, the APCs are derived from cells from a blood sample from a subject. In some embodiments, the sample is a whole blood sample. In some embodiments, the sample is an apheresis sample. In some specific embodiments, B cells are used as a source of APCs and are generated from a patient apheresis, e.g., a tumor fragment and / or an apheresis autologous to the subject from which the T cells were obtained. In some embodiments, the B cells are expanded ex vivo from a sample from a subject. In some embodiments, the B cells are cultured for expansion with an additive that promotes expansion. In some embodiments, the culture of the B cells includes the addition of IL-21, which, in some aspects, can improve the cell yield and / or viability of the expanded T cells. In some embodiments, the addition of IL-21 can shorten the period of expansion and improve the yield and / or viability of ex vivo expanded B cells.

[0214] In other specific embodiments, monocyte-derived dendritic cells are used as a source of APCs and are generated from monocytes derived from a patient apheresis, e.g., tumor fragments and / or apheresis autologous to the subject from which the T cells were obtained.

[0215] In some embodiments, isolated or generated APCs are collected and cryopreserved. Cryopreservation after APC isolation or generation provides an intermediate storage step, which can be used to time neoepitope identification and peptide generation, as described in Section IA, and / or initial T cell expansion, as described in Section IB. In some embodiments, for cryopreservation, isolated or generated APCs 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.

[0216] In some embodiments, cryopreserved cells are prepared for subsequent processing by thawing. In some cases, cells may be ready for subsequent culture with T cells and peptide immediately after thawing after one or more washing steps.

[0217] In some embodiments, APCs (e.g., PBMCs, B cells, or monocyte-derived DCs) are contacted with a pool of peptides, which may represent many different variant amino acid sequences, e.g., 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, or 100 peptides, or any value between any of the foregoing.

[0218] Peptides or peptide pools are loaded onto antigen-presenting cells (eg, dendritic cells or B cells) such as by peptide pulsing, at a concentration suitable for their presentation on the surface of the major histocompatibility complex (MHC).

[0219] In some embodiments, the peptide concentration corresponding to an individual or single peptide in a pool of peptides ranges from at or about 0.1 μg / mL to at or about 1 μg / mL, or from at or about 1 μg / mL to at or about 10 μg / mL. In some embodiments, the concentration corresponding to an individual or single peptide can be at or about 0.01 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 2.5 μg / mL, 5 μg / mL, 10 μg / mL, or any value between any of the foregoing.

[0220] In one embodiment, inducing an APC (e.g., a B cell or monocyte-derived DC) to present a mutant amino acid sequence includes introducing a nucleotide sequence encoding the mutant amino acid sequence into the APC. The nucleotide sequence is introduced into the APC so that the APC expresses and presents the mutant amino acid sequence bound to an MHC molecule on the cell membrane. The nucleotide sequence encoding the mutant amino acid can be RNA or DNA. Introduction of the nucleotide sequence into the APC can be carried out by any of a variety of different methods. Non-limiting examples of techniques useful for introducing a nucleotide sequence into an APC include transformation, transduction, transfection, and electroporation. In some cases, a peptide for binding to an MHC class II-restricted molecule is presented as a gene encoding the mutant DNA and electroporated into an antigen-presenting cell. This DNA is then in vitro transcribed into RNA encoding the peptide on the surface for recognition by CD4+ cells. In some cases, this can be done for MHC class II restricted molecules using the Tandem Mini Gene method, see, for example, published PCT patent application WO2016 / 053338 and Parkhurst et al. (2016) Clin Cancer Res., 23:2491-505. In embodiments where multiple genes are identified, the method can include preparing multiple nucleotide sequences, each encoding a mutant amino acid sequence encoded by a different gene, and introducing each nucleotide sequence into different populations of APCs. In this regard, multiple populations of APCs can be obtained, each of which expresses and presents a different mutant amino acid sequence. For example, when using tandem minigenes, APCs (e.g., B cells or monocyte-derived DCs) are electroporated with a mixture of DNAs (multiple DNAs) encoding different mutant amino acid sequences, which are then in vitro transcribed into RNA encoding peptides for surface recognition by CD4+ T cells. In some embodiments, APCs (eg, B cells or monocyte-derived DCs) are electroporated using the Lonza 4D Nucleofector sequential electroporation system.

[0221] The method includes adding T cells (e.g., from a patient with a tumor) with a culture of APCs that present the peptide, and co-culturing the APCs and T cells for a period of time to allow presentation and recognition of the peptide on the surface of the APCs by one or more T cells in the population. In provided embodiments, the T cells comprise a stimulated T cell population.

[0222] In some embodiments, APCs are first contacted or incubated with a peptide (also referred to as "loading" or "pulsing" the peptide) to prepare peptide-presenting APCs. In some embodiments, APCs (e.g., B cells or monocyte-derived DCs) are incubated with peptide for 2 hours or about 2 hours to 48 hours or about 48 hours, e.g., 2 hours or about 2 hours to 36 hours or about 36 hours, 2 hours or about 2 hours to 24 hours or about 24 hours, 2 hours or about 2 hours to 24 hours or about 24 hours, 2 hours or about 2 hours to 18 hours or about 18 hours, 2 hours or about 2 hours to 12 hours or about 12 hours, 2 hours or about 2 hours to 6 hours or about 6 hours, 6 hours or about 6 hours to 48 hours or about 48 hours, 6 hours or about 6 hours to 36 hours or about 36 hours, 6 hours or about 6 hours to 24 hours or about 24 hours, 6 hours or about 6 hours to 24 hours or about 24 hours, 6 hours or about 6 hours to 6 hours The incubation period is from 18 hours or about 18 hours, 6 hours or about 6 hours to 12 hours or about 12 hours, 12 hours or about 12 hours to 48 hours or about 48 hours, 12 hours or about 12 hours to 36 hours or about 36 hours, 12 hours or about 12 hours to 24 hours or about 24 hours, 12 hours or about 12 hours to 18 hours or about 18 hours, 18 hours or about 18 hours to 48 hours or about 48 hours, 18 hours or about 18 hours to 36 hours or about 36 hours, 18 hours or about 18 hours to 24 hours or about 24 hours, 24 hours or about 24 hours to 48 hours or about 48 hours, 24 hours or about 24 hours to 36 hours or about 36 hours, or 36 hours or about 36 hours to 48 hours or about 48 hours.In some embodiments, APCs (e.g., B cells or monocyte-derived DCs) are incubated with peptides for at or about 4 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, or any value in between. In some embodiments, APCs (e.g., PBMCs, B cells, or monocyte-derived DCs) are incubated with peptides overnight, e.g., for 8-12 hours or about 8-12 hours. In some embodiments, the co-culture incubation lasts for at or about 6 hours.

[0223] In some embodiments, T cells (e.g., stimulated T cells) and APCs (e.g., B cells or monocyte-derived DCs) can be present in culture at a T cell to APC ratio of 1:100 to 100:1, e.g., 1:50 to 50:1, 1:25 to 25:1, 1:10 to 10:1, or 1:5 to 5:1. In some embodiments, the ratio of T cells (e.g., stimulated T cells) to APCs is at or about 1:100, 1:50, 1:25, 1:10, 1:5, 1:2.5, 1:1, 2:5:1, 5:1, 10:1, 25:1, 50:1, 100:1, or any value between any of the foregoing. In some embodiments, the ratio of T cells (e.g., stimulated T cells) to APCs is 20:1 to 1:1, 15:1 to 1:1, 10:1 to 1:1, 5:1 to 1:1, or 2.5:1 to 1:1. In some embodiments, the ratio of T cells (e.g., stimulated T cells) to APCs is 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 certain embodiments, co-culture is performed by mixing T cells, e.g., a stimulated T cell population, with APCs (e.g., B cells or monocyte-derived DCs) at a ratio of about 3:1. In some embodiments, co-culture is performed by mixing T cells, e.g., a stimulated T cell population, with APCs (e.g., B cells or monocyte-derived DCs) at a ratio of about 1:1.

[0224] In some embodiments, recombinant IL-2 is present in the cell culture medium, hi some embodiments, the recombinant IL-2 is added at a concentration of 100 IU / mL to 1000 IU / mL. In some embodiments, the recombinant IL-2 is at a concentration of from 10 IU / mL or about 10 IU / mL to 600 IU / mL or about 600 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 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 to 50 IU / mL or about 50 IU / mL, from 50 IU / mL or about 50 IU / mL to 400 IU / mL or about 400 IU / mL, In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of from 50 IU / mL or about 50 IU / mL, 200 IU / mL or about 200 IU / mL, 50 IU / mL or about 50 IU / mL, 100 IU / mL or about 100 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, 400 IU / mL or about 400 IU / mL, 400 IU / mL or about 400 IU / mL, and 600 IU / mL or about 600 IU / mL. In some embodiments, recombinant IL-2 is present in an amount of 300 IU / mL or about 300 IU / mL.

[0225] In some embodiments, the co-culture of APCs and T cells can be incubated at a temperature suitable for presentation of peptides on MHC and activation of the T cells in the culture, e.g., at least about 25°C, generally at least about 30°C, generally at or about 37°C.

[0226] In some embodiments, co-culture incubation of peptide-presenting APCs with T cells is carried out for up to 96 hours. Co-culture incubation of peptide-presenting APCs can be carried out for 12 to 96 hours, e.g., at or about 12 hours, 16 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, 84 hours, or 96 hours, or any time in between. In some embodiments, the co-culture is incubated for 12 to 48 hours. In some embodiments, the co-culture is incubated for 24 to 48 hours. In certain embodiments, the co-culture is incubated for 20 to 24 hours. In some embodiments, the co-culture is incubated for about 20 hours. In some embodiments, the co-culture is incubated for about 24 hours.

[0227] In some embodiments, the method includes co-culturing the second T cell population with autologous antigen-presenting cells (APCs) for about 12 to 48 hours with recombinant IL-2 added at a concentration of 100 IU / mL to 1000 IU / mL to generate a third T cell population, where the APCs are loaded with a pool of tumor-derived peptide neoantigens, each peptide being 13 to 40 amino acids in length and loaded at a concentration of 100 ng / mL per peptide, at a ratio of the second T cell population to APCs of about 2:1 to 10:1. In some embodiments, the recombinant IL-2 is added at about 300 IU / mL. In some embodiments, the co-culturing of the T cells with APCs is performed for about 20 hours at a T cell to APC ratio of about 5:1, with IL-2 added at about 300 IU / mL. In some embodiments, co-culture of T cells with APCs is performed at a T cell to APC ratio of about 5:1 with recombinant IL-2 added at about 300 IU / mL for about 24 hours. In some embodiments, the pool of peptide neo-antigens comprises up to 200 peptides, e.g., at least 100 peptides, at least 110 peptides, at least 120 peptides, at least 130 peptides, at least 140 peptides, at least 150 peptides, at least 160 peptides, at least 170 peptides, at least 180 peptides, at least 190 peptides, or at least 200 peptides, or any value in between any of the foregoing. In some embodiments, the peptide pool comprises about 190 peptides.

[0228] In some embodiments, at the end of the co-culture, tumor-reactive T cells are separated from APCs present in the co-culture. In some embodiments, the separation can include a method for selecting off or eliminating APCs. In some embodiments, the separation can include a method for positively selecting or retaining T cells present in the co-culture. In some embodiments, the total T cells in the co-culture can be selected. In certain embodiments, tumor-reactive T cells or T cells expressing one or more upregulated markers, e.g., activation markers, associated with tumor-reactive T cells can be selected. Exemplary methods for selecting tumor-reactive T cells are described in Section II.D.

[0229] D. Selection of Tumor-Reactive T Cells In embodiments of the provided methods, prior to further expansion of T cells from the co-culture, the method includes enriching or selecting tumor-reactive T cells that are surface-positive for CD134 (OX40) and / or CD137 (41BB). The selected cells that are surface-positive for CD134 and / or CD137 are a fourth cell population according to the provided methods. Cell selection can be by antibody binding to CD134 and / or CD137 markers and subsequent enrichment by methods including flow cytometry, e.g., magnetic separation or fluorescence-activated cell sorting (FACS). In certain embodiments, the method includes selecting cells from the co-culture for cells that are surface-positive for CD134 and CD137 to generate a fourth cell population (third cell population). In some embodiments, the T cells selected from the co-culture provide a T cell population enriched for CD3+ T cells or CD4+ and CD8+ cells that are positive for one or more of such T cell activation markers CD134 and / or CD137, e.g., CD134 and CD137. In some embodiments, such cells include tumor-reactive T cells or are enriched for tumor-reactive T cells. For example, such CD3+ T cells, or CD4 + and / or CD8 +The population can be further sorted into subpopulations by positive selection for CD134 and / or CD137 markers, which are expressed or relatively highly expressed on tumor-reactive T cells. In certain embodiments, selection of cells from the co-culture generates a fourth cell population, which is an enriched cell population for further culture under expansion conditions as described in Section II.E.

[0230] Methods for isolating, selecting, and / or enriching cells can be by any of a variety of methods, for example, methods based on positive or negative selection. In some embodiments, the methods can include immunoaffinity-based selection. In some embodiments, T cells can be enriched or sorted by a variety of methods, including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed-cartridge-based cell sorters. In certain aspects, CD134 and / or CD137 can be used to select reactive cells using, but not limited to, fluorescent antibodies, nanoparticles, or beads on cell sorting equipment, including, but not limited to, CliniMACS, Sony FX500, or Tyto cell sorting systems (Miltenyi).

[0231] In some embodiments, T cells can be enriched or sorted by a variety of methods, including, but not limited to, magnetic bead separation, fluorescent cell sorting, and disposable closed cartridge-based cell sorters. In certain aspects, one or more reagents specific for CD134 and / or CD137 can be used, including, but not limited to, fluorescent antibodies, nanoparticles, or beads on cell sorting equipment, including, but not limited to, CliniMACS, Sony FX500, or Tyto cell sorting systems (Miltenyi).

[0232] In certain embodiments, the sample is contacted with a binding agent, e.g., a detectably labeled binding agent, that specifically binds to the cell surface markers CD134 and / or CD137. In certain embodiments, the detectably labeled binding agent is fluorescently labeled. In certain embodiments, T cells labeled with the binding agent specific for the cell surface markers are identified by flow cytometry. In certain embodiments, the method further includes separating any resulting T cells labeled with the binding agent from other components of the sample to produce a composition enriched for T cells that are surface-positive for one or more cell surface markers. Cell sorting equipment with sufficiently high throughput to handle large volumes and cell numbers can be used. Non-limiting examples of cell sorting equipment include, for example, the Sony FX500 or the Tyto cell sorting system (Miltenyi).

[0233] Incubation is generally carried out under conditions in which the antibody or binding partner, or a molecule, e.g., a secondary antibody or other reagent that specifically binds to such an antibody or binding partner attached to a magnetic particle or bead and / or detectably labeled, specifically binds to a cell surface molecule if present on cells in the sample. In some aspects, antibody-bound cells can be recovered or separated from unbound cells in the sample.

[0234] In some aspects, the separation is performed as an automated separation of cells in a closed and sterile system. For example, the components of such an automated system may include an integrated microcomputer, a fluorescent light source and separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the integrated computer controls all components of the instrument and commands the system to perform repetitive procedures in a unified sequence.

[0235] In some embodiments, antibodies that bind to cell surface markers CD134 and CD137 are each bound to magnetic beads. In such embodiments, the cells are incubated with the magnetically labeled beads, and then the magnetic beads are removed or separated. In some embodiments, the magnetic separation unit includes a movable permanent magnet and a holder for the selection column. In some aspects, separation is performed using a CliniMACS system (Miltenyi Biotic) or any similar system known in the art. In certain embodiments, separation is performed using a system with a cell processing unit that automatically washes the cells and fractionates them by centrifugation. In some embodiments, automated separation using, for example, the CliniMACS system, uses antibody-bound particles provided in a sterile solution. In some embodiments, cells are labeled with detectable particles, and then the cells are washed to remove excess particles. After initiating the computerized separation program, the system automatically applies the cell sample to the separation column. Antibody-labeled cells are retained in the column, while unlabeled cells are removed by a series of washing steps. In some aspects, cell populations for use with the methods described herein are labeled, retained in a column, eluted from the column after the magnetic field is removed, and collected in a cell collection bag.

[0236] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some examples, cells are separated based on binding of an antibody or other binding partner specific for one or more cell surface markers, and are transported in a fluid stream, for example, by fluorescence-activated cell sorting (FACS), including preparative-scale (FACS), coupled with a flow cytometry detection system, and / or a microelectromechanical system (MEMS) chip.

[0237] In certain embodiments, cell selection is performed by flow cytometry-based cell sorting. Compared to other methods, flow cytometry-based cell sorting has the advantage of isolating cells in a single step based on multiple parameters of each cell, thereby achieving higher cell yields and higher purity that may not be possible with bead-based (e.g., magnetic bead-based) separation. Furthermore, multiparameter cell staining and separation allows for simultaneous labeling, identification, and sorting of multiple antigens and characteristic fluorescent signals. Using flow cytometry sorting, specific populations can be removed and isolated based on complex cell surface phenotypes in a single process. Cell sorting equipment with sufficiently high throughput to handle large volumes and cell numbers can be used. Non-limiting examples of cell sorting equipment include the Sony FX500 or Tyto Cell Sorting System (Miltenyi). For use in the provided methods, the flow cytometer equipment is GMP-compliant. Cell sorting methods to achieve multiparameter sorting for two or more cell surface markers (e.g., CD134 and CD137) can be performed using compatible multicolor fluorophore reagents. It is within the level of ordinary skill in the art to select appropriate fluorophores and reagents, for example, by selecting bright fluorophores and fluorophores with minimal or no spectral overlap. In some embodiments, the one or more fluorescent markers each individually comprise a fluorophore selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

[0238] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some examples, cells are separated based on the binding of an antibody or other binding partner specific to one or more cell surface markers, and then transported into a fluid stream, for example, by fluorescence-activated cell sorting (FACS), including a preparative scale (FACS), and / or a microelectromechanical system (MEMS) chip, combined with a flow cytometry detection system. In some embodiments, the cell populations described herein are collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers (e.g., by antibody-conjugated fluorescent peptides) are transported into a fluid stream.

[0239] In some embodiments, cell staining involves incubation with an antibody or binding partner that specifically binds to a marker as described, in some embodiments, followed by a washing step and separating cells bound to the antibody or binding partner from cells that are not bound to the antibody or binding partner. In some aspects of such processes, a volume of cells is mixed with an amount of the desired staining reagent and incubated under conditions for staining the cells. In some embodiments, staining or labeling is performed at a temperature between 0°C and 25°C, e.g., at or about 4°C. In some embodiments, staining or labeling is performed for more than 5 minutes, typically more than 15 minutes. In some embodiments, staining or labeling is performed for 15 minutes to 6 hours, e.g., 30 minutes to 2 hours. In some embodiments, staining or labeling is performed for, e.g., 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any value in between any of the foregoing. In some embodiments, labeling with one or more staining reagents is performed simultaneously. In some embodiments, one or more wash steps are performed before introducing the sample into a flow cytometer for analysis.

[0240] In some embodiments, the cell sample is prepared at a concentration of 1 x 10 to allow the cells to pass through a flow cytometer for reading. 6 ~5×10 7 The culture medium is prepared by suspending single cells at a density of 5×10 cells / ml. In some embodiments, the density of cells for sorting is 5×10 6 cells / mL~50×10 6 cells / mL, e.g., 20 x 10 6 cells / mL or approximately 20 x 10 6 The cell sheath is typically 1000-20,000 cells / mL. In some embodiments, this concentration of cells is referred to as the fluid sheath. In some embodiments, the fluid sheath influences the speed of flow sorting, which typically proceeds at about 2,000-20,000 cells (events) per second. The fluid sheath for the cell sample is typically made from phosphate buffered saline, although other solutions are available, as known and understood by those skilled in the art.

[0241] In some embodiments, the flow cytometry sorting rate is between 2,000 events / second and 10,000 events / second. In some embodiments, the flow cytometry sorting rate is about 2,000 events / second, about 3,000 events / second, about 4,000 events / second, about 5,000 events / second, about 6,000 events / second, about 7,000 events / second, about 8,000 events / second, about 9,000 events / second, about 10,000 events / second, about 15,000 events / second, or about 20,000 events / second, or any value between any of the foregoing. In some embodiments, the flow cytometry sorting rate is about 6,000 events / second.

[0242] In some embodiments, a sample is introduced into a flow cytometer. The cell sample is typically forced into a single stream through a fluidic system using hydraulic pressure. This stream then passes through one or more beams of light scattering or fluorescence emission. In a flow cytometer, a laser typically serves as the light source. Upon contact with the cell sample, the laser generates a single wavelength of light that produces forward-scattered light as a measure of cell size, side-scattered light as a measure of cellular complexity, and side-emitted fluorescence proportional to the relative abundance of specific cell markers. Fluorescence channels are typically designated by names such as FL1, FL2, and FL3, depending on the number of channels in the instrument. Each fluorescence channel is equipped with a barrier filter to detect a specific dye of choice and reject all others. The channel in which a particular dye is primarily detectable may be referred to as its primary fluorescence channel, while other fluorescence channels may be referred to as secondary channels. The light scattering and fluorescence emission signals are processed by a flow cytometry engine and converted into electronic pulses that are displayed in a graphical user interface (GUI).

[0243] Methods for analyzing flow cytometry or FACS data can include "gating" the data to separate specific cell populations. Various cell types can be identified by the scattering parameters and fluorescence emission resulting from labeling various cellular proteins with the dye-labeled antibodies described above. Identification of clusters, and therefore populations, can be achieved by "gating" the cells. In some embodiments, a gate corresponding to a subset of particles of interest, e.g., neoantigen-reactive TIL-expressing markers, is defined by the user with the aid of software operably associated with the flow system, as described above.

[0244] In some embodiments, the gate may be a "threshold" gate, which is a gate on only one optical parameter that defines an aperture region in multidimensional space. In some embodiments, "threshold" gating can be used on forward light scatter to remove high-frequency low-level signals caused by interference such as debris in the sample. In some embodiments, "window" gating is used, for example, by defining upper and lower limits for signal values. In some embodiments, gating is performed on a 2D plot of two parameters, such as side scatter (e.g., on the vertical axis) and fluorescence signal (e.g., on the horizontal axis).

[0245] In some embodiments, flow cytometry for cell surface markers involves gating on an "F minus one" (FMO) control. FMO gating involves staining a separate portion of the same sample with a panel of detectably labeled binding agents containing all but one of the agents. The distribution of the signal of the removed fluorophore can be used to define the positivity threshold for the missing label, since all cells are known to be negative in the control. The position of the entire gate can be determined using a fluorescence minus one (FMO) control in which the antibody for the investigated marker is replaced with an appropriate isotype control. In some embodiments, a gate can be drawn using cells stained with the FMO cocktail around cells positive for CD137 and / or CD134. In some embodiments, staining with other labeled antibody reagents or scatter analysis can be performed with sequential gating to exclude other cell populations (e.g., APCs) and / or enrich for lymphocytes (e.g., CD3+ or CD4+ and / or CD8+ cells). In some embodiments, a viability dye can also be added. An exemplary viability dye is 7-ADD. In some embodiments, cells negative for 7-AAD (7-AAD neg ) you can draw a gate around it.

[0246] In some embodiments, the cells are sorted and collected into a single cell population. The selected cell population is referred to as the fourth cell population and is used as input for expansion, as described in Section II.E.

[0247] Separation need not result in the enrichment or removal of a particular cell population, or 100% of cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to an increase in the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker.

[0248] In some embodiments, the selection results in an enriched cell population that is additionally positive for CD134 and / or CD137, e.g., a cell population enriched for CD3+ T cells or CD4+ and CD8+ cells. In some embodiments, such cells comprise or are enriched for tumor-reactive T cells or T cells associated with tumor-reactive T cells. In some embodiments, the enriched cell population is used in subsequent processing steps, e.g., subsequent processing steps involving incubation, stimulation or activation, and / or expansion by one or more steps of any of the provided methods.

[0249] In some embodiments, the enriched cell population is enriched cells derived from a starting sample as described above, wherein the percentage of cells of a particular phenotype within the enriched cell population, e.g., tumor-reactive CD3+ T cells, or CD3+ T cells that are surface positive for CD134 and / or CD137, is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000% or more over the percentage of such cells in the starting sample. In some embodiments, the purity of tumor-reactive CD3+ T cells, or CD3+ T cells surface-positive for CD134 and / or CD137, in the enriched composition, i.e., the percentage of cells positive for the selected cell surface marker relative to the total cells in the enriched cell population, is at least 90%, 91%, 92%, 93%, 94%, and generally at least 95%, 96%, 97%, 98%, 99% or more.

[0250] E. Further Growth and Harvesting In some embodiments, the selected T cells from the co-culture (e.g., a fourth cell population) are further incubated under conditions to expand the cells ex vivo after co-culture. The incubation is performed in the presence of one or more T cell stimulatory agents under conditions to stimulate the T cells, e.g., to expand the T cells. In some embodiments, the incubation is a rapid expansion protocol (REP).

[0251] In some of the provided embodiments, the T cell stimulatory agent is selected from an agent that initiates TCR / CD3 intracellular signaling and / or an agent that initiates signaling through a costimulatory receptor. Anti-CD3 antibodies can include any antibody that is directed against or can specifically bind to a 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. The anti-CD3 antibody can be added as a soluble reagent or bound to beads. In certain embodiments, the anti-CD3 antibody is soluble. In some of the provided embodiments, the agent that initiates TCR / CD3 intracellular signaling is an anti-CD3 antibody, such as OKT3.

[0252] 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 , from 1 ng / mL or about 1 ng / mL, 15 ng / mL or about 15 ng / mL, 1 ng / mL or about 1 ng / mL, 5 ng / mL or about 5 ng / mL, 5 ng / mL or about 5 ng / mL, 50 ng / mL or about 50 ng / mL, 5 ng / mL or about 5 ng / mL, 30 ng / mL or about 30 ng / mL, 5 ng / mL or about 5 ng / mL, 15 ng / mL or about 15 ng / mL, 15 ng / mL or about 15 ng / mL, 50 ng / mL or about 50 ng / mL, 15 ng / mL or about 15 ng / mL, 30 ng / mL or about 30 ng / mL, or 30 ng / mL or about 30 ng / mL, 50 ng / mL or about 50 ng / mL (inclusive). In one embodiment, anti-CD3 is added to cell culture medium at 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, or about 50 ng / mL. In some embodiments, the anti-CD3 antibody is added at a concentration of about 30 ng / mL. In a specific embodiment, the anti-CD3 antibody is OKT3.

[0253] In some embodiments, the T cell stimulatory agent can include adding an anti-CD3 antibody and can further include adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), to the T cell population. In some aspects, the PBMCs provide a CD28-mediated signal to provide a costimulatory signal to the T cells. In some aspects, the non-dividing feeder cells can be irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some embodiments, the non-dividing PBMCs, such as irradiated PBMCs, are added at a ratio of PBMC to T cells that is 100:1 to 500:1. In some embodiments, the non-dividing PBMCs, such as irradiated PBMCs, are added at a ratio of PBMC to T cells that is 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, or 500:1, or any value between any of the foregoing. In some embodiments, non-dividing PBMCs, such as irradiated PBMCs, are added at a ratio of PBMCs to T cells that is about 200:1.

[0254] Generally, culturing and incubation can be carried out in the presence of recombinant IL-2. In some embodiments, recombinant IL-2 is added to the culture medium or is exogenous to the culture medium. In some embodiments, recombinant IL-2 is present 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 to 4000 IU / mL or about 4000 IU / mL, from 1000 IU / mL or about 1000 IU / mL to 2000 IU / mL or about 2000 IU / mL, from 2000 IU / mL to 8000 IU / mL or about 8000 IU / mL, from 2000 IU / mL or about 2000 IU / mL to 7000 IU / mL or about 7000 IU / mL, from 2000 IU / mL or about 2000 IU / mL to 6000 IU / mL or from about 6000 IU / mL, 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, 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, 6000 IU / mL or about 6000 IU / mL, 4000 IU / mL or about 4000 IU / mL to 5000 IU / mL or about 5000 IU / mL, 5000 IU / mL or about 5000 IU / mL to 8000 IU / mL or about 8000 IU / mL, 5000 IU / mL or about 5000 IU / mL to 7000 IU / mL, 5000 IU / mL or about 5000 IU / mL to 6000 IU / mL,The recombinant IL-2 is added to the culture medium at a concentration of 6000 IU / mL or about 6000 IU / mL to 8000 IU / mL or about 8000 IU / mL, 6000 IU / mL or about 6000 IU / mL to 7000 IU / mL or about 7000 IU / mL, or 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.

[0255] In some embodiments, selected cells from the co-culture (e.g., a fourth cell population) are expanded by culturing the T cells with irradiated PBMCs added at a ratio of 200:1 iPBMC to TILs, along with 6000 IU / mL human recombinant IL-2 and 30 ng / mL anti-CD3 antibody (OKT3).

[0256] The sorted or selected T cells can be expanded under one or more stimulatory conditions 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 can occur in a bioreactor. In some embodiments, a composition of expanded T cells is removed from the closed system and placed in and / or connected to a bioreactor for expansion. The sorted or selected T cells can be expanded using a cell expansion system, for example, by transferring the cells to a gas-permeable bag in association 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 steps of the method can be performed in a closed system or under GMP conditions.

[0260] In some embodiments, incubation with a T cell stimulator for expansion of tumor-reactive cells is carried out until a threshold number of cells is reached. In some embodiments, the threshold number of cells is about 100 times or more the number of selected TILs in the fourth T cell population before expansion. In some embodiments, the threshold number of cells is about 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 1250 times, 1500 times, or 2000 times the number of selected TILs in the fourth population before expansion, or any value therebetween.

[0261] In some embodiments, incubation with a T cell stimulator for expansion is for 7 to 21 days, e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or any value between any of the foregoing. In some embodiments, incubation is for 7 to 14 days. In some embodiments, incubation is for about 10 days. In some embodiments, incubation is for about 11 days. In some embodiments, incubation is for about 12 days. In some embodiments, incubation is for about 13 days. In some embodiments, incubation is for about 14 days. Optionally, medium can be changed daily, every other day, every third day, every fourth day, or once a week during the culture or incubation period. In some embodiments, medium changes are on days 5 and 10. In some embodiments, recombinant IL-2 is replenished (added) with each medium change. In some embodiments, about 6000 IU / mL of recombinant IL-2 is replenished (added) with each medium change.

[0262] In some of the provided embodiments, the culturing step comprises culturing 0.5 x 10 8 or approximately 0.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 Total cells or total viable cells, 0.5 x 10 8 or approximately 0.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 Total cells or total viable cells, 0.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 Total cells or total viable cells, 0.5 x 10 8 or approximately 0.5 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 Total cells or total viable cells, 0.5 x 10 8 or approximately 0.5 x 108 From 15 x 10 8 pieces or approximately 15 x 10 8 Total cells or total viable cells, 0.5 x 10 8 or approximately 0.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 Total cells or total viable cells, 0.5 x 10 8 or approximately 0.5 x 10 8 From 3.5 x 10 8 pieces or approximately 3.5 x 10 8 Total cells or total viable cells, 0.5 x 10 8 or approximately 0.5 x 10 8 From 1 x 10 8 or approximately 1 x 10 8 Total cells or total viable cells, 1 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 Total cells or total viable cells, 1 x 10 8 or approximately 1 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 Total cells or total viable cells, 1 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 Total cells or total viable cells, 1 x 10 8 or approximately 1 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 Total cells or total viable cells, 1 x 10 8 or approximately 1 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 Total cells or total viable cells, 1 x 10 8 or approximately 1 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 Total cells or total viable cells, 1 x 10 8 or approximately 1 x 10 8 From 3.5 x 10 8pieces or approximately 3.5 x 10 8 Total cells or total viable cells, 3.5 x 10 8 pieces or approximately 3.5 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 Total cells or total viable cells, 3.5 x 10 8 pieces or approximately 3.5 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 Total cells or total viable cells, 3.5 x 10 8 pieces or approximately 3.5 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 Total cells or total viable cells, 3.5 x 10 8 pieces or approximately 3.5 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 Total cells or total viable cells, 3.5 x 10 8 pieces or approximately 3.5 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 Total cells or total viable cells, 3.5 x 10 8 pieces or approximately 3.5 x 10 8 From 8 x 10 8 pieces or approximately 8 x 10 8 Total cells or total viable cells, 8 x 10 8 pieces or approximately 8 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 Total cells or total viable cells, 8 x 10 8 pieces or approximately 8 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 Total cells or total viable cells, 8 x 10 8 pieces or approximately 8 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 Total cells or total viable cells, 8 x 10 8 pieces or approximately 8 x 10 8From 60 x 10 8 or approximately 60 x 10 8 Total cells or total viable cells, 8 x 10 8 pieces or approximately 8 x 10 8 From 15 x 10 8 pieces or approximately 15 x 10 8 Total cells or total viable cells, 15 x 10 8 pieces or approximately 15 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 Total cells or total viable cells, 15 x 10 8 pieces or approximately 15 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 Total cells or total viable cells, 15 x 10 8 pieces or approximately 15 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 Total cells or total viable cells, 15 x 10 8 pieces or approximately 15 x 10 8 From 60 x 10 8 or approximately 60 x 10 8 Total cells or total viable cells, 60 x 10 8 or approximately 60 x 10 8 From 50 x 10 9 or approximately 50 x 10 9 Total cells or total viable cells, 60 x 10 8 or approximately 60 x 10 8 From 30 x 10 9 pieces or approximately 30 x 10 9 Total cells or total viable cells, 60 x 10 8 or approximately 60 x 10 8 From 12 x 10 9 pieces or approximately 12 x 10 9 Total cells or total viable cells, 12 x 10 9 pieces or approximately 12 x 10 9 From 50 x 10 9 or approximately 50 x 10 9 Total cells or total viable cells, 12 x 10 9pieces or approximately 12 x 10 9 From 30 x 10 9 pieces or approximately 30 x 10 9 total cells or total viable cells, or 30 x 10 9 pieces or approximately 30 x 10 9 From 60 x 10 9 or approximately 60 x 10 9 This is done until a threshold amount of cells is achieved, either total cells or total viable cells (inclusive).

[0263] In some of any of the provided embodiments, the method results in a fold expansion of T cells that is at or about at least 100-fold, at or about at least 250-fold, at or about at least 250-fold, at or about at least 500-fold, at or about at least 1000-fold, or more, relative to the number of cells in the fourth cell population before expansion.

[0264] Once a therapeutic dose is reached after expansion, the product can be concentrated and frozen in a cryopreservation medium. In some embodiments, the cryoprotectant is or includes DMSO and / or glycerol. In some embodiments, compositions formulated for cryopreservation can be stored at low temperatures, e.g., ultra-low temperatures, e.g., at temperatures ranging from -40°C to -150°C, e.g., at or about 80°C ± 6.0°C.

[0265] Also provided herein is a T cell population produced by the method described herein, and its pharmaceutical composition.The cell composition is enriched with tumor-reactive T cells.In some embodiments, the cell composition is characterized by one or more of the characteristics described in Section II.

[0266] III. Therapeutic Methods and Uses Provided herein are compositions and methods relating to the therapeutic cell compositions described herein for use in treating a subject's disease or condition, such as cancer. Such methods and uses include, for example, therapeutic methods and uses comprising administering therapeutic cells or compositions containing the same to a subject with a disease, condition, or disorder. In some cases, the disease or disorder is a tumor or cancer. In some embodiments, the cells or pharmaceutical compositions thereof are administered in an amount effective to treat the disease or disorder. Uses include such methods and treatments, as well as the use of the cells or pharmaceutical compositions thereof in the preparation of a medicament for performing such therapeutic methods. In some embodiments, the method thereby treats the subject's disease, condition, or disorder.

[0267] In some embodiments, the cell compositions provided herein are autologous to the subject to be treated.In such embodiments, the starting cells for expansion are directly isolated from the biological sample obtained from the subject as described herein, optionally including enrichment of surface-positive T cells for one or more T cell activation markers as described, and cultured under the conditions for expansion provided herein.In some aspects, the biological sample obtained from the subject is or comprises tumor sample or lymph node sample, and such sample tumor, and such tissue amount is obtained, for example, by resection or biopsy (for example, core needle biopsy or fine needle aspiration).In some embodiments, after culturing under the conditions for expansion by the provided method, cells are formulated and optionally cryopreserved for subsequent administration to the same subject to treat cancer.

[0268] In some embodiments, the treatment method comprises administering an effective amount of a composition containing surface CD3+ T cells, which may comprise tumor-reactive CD3+ T cells or T cells surface-positive for one or more activation markers. Such compositions may include any of those described herein, including compositions produced by the provided methods.

[0269] In some embodiments, a subject (e.g., a self-administered) is administered 10 5 pieces or about 10 5 From 10 12 pieces or about 10 12 10 CD3+ T cells, or 10 generated by any of the provided methods 5 pieces or about 10 5 From 10 8 pieces or about 10 8 10 CD3+ T cells, or 10 generated by any of the provided methods 6 pieces or about 10 6 From 10 12 pieces or about 10 12 10 CD3+ T cells, or 10 generated by any of the provided methods 8 pieces or about 10 8 From 10 11 pieces or about 10 11 10 CD3+ T cells, or 10 generated by any of the provided methods 9 pieces or about 10 9 From 10 10 pieces or about 10 10 In some embodiments, the therapeutically effective amount for administration is 10 CD3+ T cells produced by any of the provided methods. 5 More than 10 5 More than or about 10 5 More than 10 CD3+ T cells, generated by any of the provided methods 6 pieces or about 10 6 10 CD3+ T cells, generated by any of the provided methods 7 pieces or about 10 7 10 CD3+ T cells, generated by any of the provided methods 8 pieces or about 10 8 10 CD3+ T cells, generated by any of the provided methods 9 pieces or about 10 9 10 CD3+ T cells, generated by any of the provided methods 10 pieces or about 1010 10 CD3+ T cells, generated by any of the provided methods 11 pieces or about 10 11 10 CD3+ T cells, or 10 generated by any of the provided methods 12 pieces or about 10 12 In some embodiments, such an amount can be administered to a subject with a disease or condition, such as a cancer patient. In some embodiments, the number of T cells administered are viable T cells.

[0270] In some embodiments, the treatment method comprises administering an effective amount of a composition containing tumor-reactive CD3+ T cells or CD3+ T cells that are surface-positive for one or more activation markers. Such compositions can include any of those described herein, including compositions produced by the provided methods. In some embodiments, 10 5 pieces or about 10 5 From 10 12 pieces or about 10 12 tumor-reactive CD3+ T cells, or CD3+ T cells surface positive for one or more activation markers such as any of those described, or 10 5 pieces or about 10 5 From 10 8 pieces or about 10 8 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, or 10 6 pieces or about 10 6 From 10 12 pieces or about 10 12 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, or 10 8 pieces or about 10 8 From 10 11 pieces or about 10 11 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, or 10 9 pieces or about 109 From 10 10 pieces or about 10 10 In some embodiments, the therapeutically effective amount for administration is 10 to 1000 tumor-reactive CD3+ T cells or CD3+ T cells that are surface positive for one or more activation markers. 5 More than 10 5 More than or about 10 5 >10 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers 6 pieces or about 10 6 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, 10 7 pieces or about 10 7 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, 10 8 pieces or about 10 8 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, 10 9 pieces or about 10 9 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, 10 10 pieces or about 10 10 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, 10 11 pieces or about 10 11 tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more activation markers, or 10 12 pieces or about 10 12 The amount of T cells administered includes tumor-reactive CD3+ T cells or CD3+ T cells that are surface-positive for one or more activation markers. In some embodiments, such an amount can be administered to a subject with a disease or condition, such as a cancer patient. In some embodiments, the number of T cells administered is viable T cells.

[0271] In some embodiments, the amount is administered as a flat dose, hi other embodiments, the amount is administered per kilogram of the subject's body weight.

[0272] In some embodiments, for example, compositions containing tumor-reactive T cells or T cells surface-positive for T cell activation markers, generated by any of the provided methods, are administered to an individual immediately after expansion by the provided methods. In other embodiments, the expanded T cells, for example, expanded tumor-reactive T cells or expanded T cells surface-positive for T cell activation markers, are cryopreserved, for example, by the methods described above, before administration. For example, T cells, for example, tumor-reactive T cells or T cells surface-positive for T cell activation markers, can be stored for more than 6, 12, 18, or 24 months before administration to an individual. Such cryopreserved cells can be thawed before administration.

[0273] In some embodiments, for example, a provided composition provided by any of the provided methods or containing tumor-reactive T cells or T cells surface-positive for a T cell activation marker can be administered to a subject by any convenient route, including parenteral routes, e.g., subcutaneous, intramuscular, intravenous, and / or epidural administration routes.

[0274] In some embodiments, for example, a composition provided by any of the provided methods, or containing tumor-reactive T cells or T cells surface-positive for a T cell activation marker, can be administered in a single dose. Such administration can be by injection, for example, intravenous injection. In some embodiments, tumor-reactive T cells or T cells surface-positive for a T cell activation marker can be administered in multiple doses. Administration can be once, twice, three times, four times, five times, six times, or more than six times per year. Administration can be once per month, once every two weeks, once per week, or once every other day. Administration of such compositions and cells can continue as long as necessary.

[0275] In some embodiments, a subject is administered lymphocyte depletion therapy before receiving a dose of cells, e.g., produced by any of the provided methods, or from a provided composition containing tumor-reactive T cells or T cells surface-positive for a T cell activation marker. Lymphocyte depletion therapy can include administration of fludarabine and / or cyclophosphamide (the active form is called mafosfamide) and combinations thereof. Such methods are described in Gassner et al. (Cancer Immunol Immunother. 2011, 60(1):75-85; Muranski, et al., Nat Clin Pract Oncol, 2006, 3(12):668-681; Dudley, et al., J Clin Oncol 2008, 26:5233-5239; and Dudley, et al., J Clin Oncol. 2005, 23(10):2346-2357. In some embodiments, fludarabine is administered at a dose of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day, or a dose between any of the foregoing ranges. In some embodiments, fludarabine is administered for 2 to 7 days, e.g., 3 to 5 days, e.g., 3 days or about 3 days, 4 days or about 4 days, or 5 days or about 5 days. In some embodiments, cyclophosphamide is administered for 2 to 7 days, e.g., 3 to 5 days, e.g., 3 days or about 3 days, 4 days or about 4 days, or 5 days or about 5 days. Cyclophosphamide is administered at a dosage of 100 mg / m² / day, 150 mg / m² / day, 175 mg / m² / day, 200 mg / m² / day, 225 mg / m² / day, 250 mg / m² / day, 275 mg / m² / day, or 300 mg / m² / day. In some embodiments, cyclophosphamide is administered intravenously (i.e., iv). In some embodiments, cyclophosphamide treatment lasts for 2-7 days, e.g., 3-5 days, 3 days or about 3 days, 4 days or about 4 days, or 5 days or about 5 days. Lymphocyte depletion therapy is administered prior to the provided cell composition.In some embodiments, lymphocyte depletion therapy is administered within one week of administration of a provided cell composition, for example, 5-7 days prior to administration of a dose of cells.

[0276] The compositions described herein can be used in methods for treating hyperproliferative disorders. In a preferred embodiment, they are used to treat cancer. In some aspects, cancer can be melanoma, ovarian cancer, cervical cancer, lung cancer, bladder cancer, breast cancer, head and neck cancer, renal cell carcinoma, acute myeloid leukemia, colorectal cancer and sarcoma. In some embodiments, cancer is a cancer with a high mutation burden. In some embodiments, 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.

[0277] In some embodiments, the cancer is an epithelial cancer. In some embodiments, the cancer is selected from non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, and endometrial cancer. In some embodiments, the breast cancer is HR+ / Her2- breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the breast cancer is HER2+ breast cancer.

[0278] In some embodiments, the subject has a cancer that is a hematological tumor. Non-limiting examples of hematological tumors include leukemia, such as acute leukemia (such as l lq23-positive acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and aggressive), multiple myeloma, Waldenstroth hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0279] In some embodiments, the subject has a solid tumor cancer. Non-limiting examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid tumors, pancreatic cancer, breast cancer (including basal breast cancer, ductal carcinoma, and lobular carcinoma of the breast), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, thyroid cancer, and thyroid cancer. These include head cancer, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some examples, the tumor is melanoma, lung cancer, lymphoma, breast cancer, or colon cancer.

[0280] In some embodiments, the cancer is skin cancer. In particular embodiments, the cancer is melanoma, such as cutaneous melanoma. In some embodiments, the cancer is Merkel cell carcinoma or metastatic cutaneous squamous cell carcinoma (CSCC).

[0281] In some embodiments, the tumor is a carcinoma, which is a cancer that arises from epithelial cells or is of epithelial origin. In some embodiments, the cancer arises from epithelial cells, including, but not limited to, breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cancer, esophageal cancer, small intestine and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer and skin cancer, e.g., squamous cell carcinoma and basal cell cancer, prostate cancer, renal cell carcinoma, and other known cancers that affect epithelial cells throughout the body.

[0282] In some embodiments, the subject has a cancer of the gastrointestinal (GI tract), for example, cancer, or a gastrointestinal cancer that includes the upper or lower gastrointestinal tract, or accessory organs of digestion, such as the esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum, or anus. In some embodiments, the cancer is esophageal cancer, gastric cancer (gastric cancer), pancreatic cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, cancer of mucosa-associated lymphoid tissue (MALT lymphoma), cancer of the bile duct, colorectal cancer (including colon cancer, rectal cancer, or both), anal cancer, or gastrointestinal carcinoid tumor. In certain embodiments, the cancer is colorectal cancer.

[0283] In some embodiments, cancer is colorectal cancer (CRC).Colorectal cancer (CRC) is a common tumor that is increasing in incidence, and often does not respond to checkpoint blockade or other immunotherapy.This is true even if such cancer has characteristics related to response, such as a certain high mutation rate, and the well-established correlation between prognosis and the level of T cell infiltration.

[0284] In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is triple-negative breast cancer (TNBC).

[0285] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is Merkel cell carcinoma. In some embodiments, the cancer is metastatic cutaneous squamous cell carcinoma (CSCC). In some embodiments, the cancer is melanoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC).

[0286] In some embodiments, the subject is one whose cancer is resistant to checkpoint blockade, such as anti-PD1 therapy or anti-PD-L1 therapy, or has relapsed after treatment with such checkpoint blockade.

[0287] In some embodiments, the subject is the same subject from whom the biological sample was obtained to generate the therapeutic cell composition. In some such embodiments, the method of treatment provided is adoptive cell therapy with a therapeutic composition containing T cells autologous to the subject.

[0288] In some embodiments, the cell compositions provided herein are allogeneic to the subject to be treated. In some aspects, the subject from which the cells are derived or isolated is a healthy subject or is not known to have a disease or condition, such as cancer. In such embodiments, the starting cells for expansion are directly isolated from a biological sample from such a subject as described herein, optionally including enrichment of surface-positive T cells for one or more of the T cell activation markers described, and cultured under the expansion conditions provided herein. In some aspects, the biological sample from the subject is or includes a tumor sample or lymph node sample, and such a sample tumor, and a quantity of such tissue is obtained, for example, by resection or biopsy (e.g., core needle biopsy or fine needle aspiration). In some embodiments, after culturing under expansion conditions, the cells are formulated and optionally cryopreserved for subsequent administration to a different subject to treat cancer in such a different subject.

[0289] In some embodiments, the provided methods can be performed with one or more other immunotherapies. In some embodiments, the immunotherapy is an immunomodulatory agent that is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically binds to a molecule selected from among CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, CD137 (4-1BB), GITR, CD40, CD40L, CD134 (OX40), OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, TIGIT, and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a small molecule, or a polypeptide. In some embodiments, the immune checkpoint inhibitor is selected from among nivolumab, pembrolizumab, pidilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, urelumab, TRX518, dacetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or an antigen-binding fragment thereof.

[0290] In some embodiments, the methods provided include combination therapy of the described cell therapy with a PD-1 or PD-L1 inhibitor, which may include a binding antibody, antagonist, or inhibitor (i.e., a blocking agent).

[0291] In one embodiment, the PD-I inhibitor is nivolumab (commercially available as OPDIVO from Bristol-Myers Squibb Co.), or a biosimilar, antigen-binding fragment, conjugate, or variant thereof. Nivolumab is a fully human IgG4 antibody that blocks the PD-I receptor. In one embodiment, the anti-PD-I antibody is an immunoglobulin G4κ, anti-(human CD274) antibody. Nivolumab has been assigned Chemical Abstracts Service (CAS) registration number 946414-94-4 and is also known as 5C4, BMS-936558, IDX-1106, and ONO-4538. The preparation and properties of nivolumab are described in U.S. Patent No. 8,008,449 and International Patent Publication No. WO2006 / 121168.

[0292] In another embodiment, the PD-1 inhibitor comprises pembrolizumab (commercially available as KEYTRUDA from Merck & Co., Inc., Kenilworth, NJ, USA), or an antigen-binding fragment, conjugate, or variant thereof. Pembrolizumab has been assigned CAS Registry Number 1374853-91-4 and is also known as lambrolizumab, MK-3475, and SCH-900475. The properties, uses, and preparation of pembrolizumab are described in International Patent Publication No. WO2008 / 156712, U.S. Patent No. 8,354,509, and U.S. Patent Application Publication Nos. US2010 / 0266617, US2013 / 0108651, and US2013 / 0109843.

[0293] In one embodiment, the PD-LI inhibitor is durvalumab, also known as MEDI4736 (commercially available from Mediimmune, LLC, Gaithersburg, JV, a subsidiary of AstraZeneca plc.), or an antigen-binding fragment, conjugate, or variant thereof. In one embodiment, the PD-LI inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013 / 0034559.

[0294] In one embodiment, the PD-LI inhibitor is avelumab, also known as MSB0010718C (commercially available from Merck KGaA / EMD Serono), or an antigen-binding fragment, conjugate, or variant thereof. The preparation and properties of avelumab are described in U.S. Patent Application Publication No. US2014 / 0341917.

[0295] In one embodiment, the PD-LI inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (commercially available as TECENTRIQ from Genentech, Inc., a subsidiary of Roche Holding AG, Basel, Switzerland), or an antigen-binding fragment, conjugate, or variant thereof. In one embodiment, the PD-LI inhibitor is an antibody disclosed in U.S. Patent Application Publication No. 2010 / 0203056, U.S. Patent Application Publication No. 2013 / 0045200, U.S. Patent Application Publication No. 2013 / 0045201, U.S. Patent Application Publication No. 2013 / 0045202, or U.S. Patent Application Publication No. 2014 / 0065135. The preparation and properties of atezolizumab are described in U.S. Patent No. 8,217,149.

[0296] V. Kits and Articles of Manufacture Provided herein are articles of manufacture and kits that include the provided compositions, e.g., compositions comprising T cells generated by any of the provided methods, or compositions containing or enriched for tumor-reactive T cells. In some embodiments, the compositions are generated by any of the provided methods.

[0297] The kit can optionally include one or more components, such as instructions for use, equipment, and additional reagents (e.g., sterile water or saline for diluting compositions and / or reconstituting lyophilized proteins), as well as components such as tubes, containers, and syringes for carrying out the method. In some embodiments, the kit can further include reagents for sample collection, sample preparation and processing, and / or reagents for quantifying the amount of one or more surface markers in a sample, including, but not limited to, detection reagents, such as antibodies, buffers, substrates for enzyme staining, chromogens, or other materials, such as slides, containers, microtiter plates, and, optionally, instructions for carrying out the method. Those skilled in the art will recognize many other possible containers, plates, and reagents that can be used in accordance with the provided methods.

[0298] In some embodiments, the kit may be provided as an article of manufacture including packaging materials for packaging cells, antibodies, or reagents, or compositions thereof, or one or more other components. For example, the kit may include containers, bottles, tubes, vials, and any packaging materials suitable for separating or organizing the components of the kit. The container or containers may be formed from various materials, such as glass or plastic. In some embodiments, the container or containers hold a composition including cells, antibodies, or other reagents for use in the method. The article of manufacture or kit herein may include cells, antibodies, or reagents in separate containers or in the same container.

[0299] In some embodiments, the one or more containers holding the composition may be single-use or multi-use vials, and in some cases, may allow for repeated use of the composition. In some embodiments, the article of manufacture or kit may further include a second container containing a suitable diluent. The article of manufacture or kit may further include other materials desirable from a commercial, therapeutic, and user perspective, including other buffers, diluents, filters, needles, syringes, therapeutic agents, and / or package inserts with instructions for use.

[0300] In some embodiments, the kit can optionally include instructions. The instructions typically include specific language describing the cell composition, optionally other components included in the kit, and methods for using such. In some embodiments, the instructions indicate methods for using the cell composition, e.g., for administration to a subject to treat a disease or condition according to any of the provided embodiments. In some embodiments, the instructions are provided as a label or package insert on or associated with the container. In some embodiments, the instructions may provide directions for reconstituting and / or using the composition.

[0301] VI. Definition Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0302] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that aspects and variations described herein include "consisting of" and / or "consisting essentially of" aspects and variations.

[0303] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to specifically disclose all possible subranges and individual numerical values ​​within that range. For example, when a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the claimed subject matter. This applies regardless of the breadth of the range.

[0304] The term "about" as used herein refers to the normal error range of each value, which is readily known to those skilled in the art. For example, in various embodiments, about may refer to ±10%, 5%, 2.5%, or 1% of the listed nominal value. Reference to "about" any value or parameter herein also includes (and describes) aspects that are directed to the value or parameter itself. For example, a statement that refers to "about X" includes the statement "X."

[0305] As used herein, the term "autologous" refers to cells or tissues that are removed from the same organism into which they are subsequently injected or adoptively transferred.

[0306] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. The composition may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0307] As used herein, "any" or "optional" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where said event or circumstance occurs and cases where it does not occur. For example, an optionally substituted group means that the group is unsubstituted or substituted.

[0308] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammalian subjects, often humans. Pharmaceutical compositions typically contain an effective amount of an active agent (e.g., tumor-reactive T cells, such as those expanded according to the provided method) and a carrier, excipient, or diluent. Carriers, excipients, or diluents are typically pharmaceutically acceptable carriers, excipients, or diluents, respectively.

[0309] "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid or carrier conventional in the art for use with therapeutic agents that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to a recipient at the dosage and concentration used and is compatible with the other ingredients of the formulation. A pharmaceutically acceptable carrier is appropriate for the formulation being used.

[0310] As used herein, a "subject" is a mammal, such as a human or other animal, typically a human. The subject may be male or female and may be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

[0311] The term "effective amount" or "therapeutically effective amount" refers to the amount and / or concentration of a therapeutic composition, such as, for example, containing cells expanded according to the provided methods, that, when administered to a patient, results in any manner in which the symptoms of a condition, disorder, or disease or other indication are improved or otherwise beneficially altered. An effective amount for treating a disease or disorder can be an amount that relieves, reduces, or alleviates at least one symptom or biological response or effect associated with the disease or disorder, prevents the progression of the disease or disorder, or improves the patient's physical function. In certain aspects, there is a statistically significant inhibition of disease progression, for example, by improving or eliminating the symptoms and / or cause of the disease. In the case of cell therapy, an effective amount is an effective dose or number of cells administered to a patient. In some embodiments, the patient is a human patient.

[0312] As used herein, a "disease," "disorder," or "condition" refers to a pathological condition in an organism that results from a cause or condition, including, but not limited to, an infection, an acquired condition, or a genetic condition, and is characterized by identifiable symptoms. In particular, it is a condition for which treatment is required and / or desired.

[0313] As used herein, the terms "treating" a disease or disorder, "treatment" or "therapy" of a disease or disorder means slowing, halting, or reversing the progression of the disease or disorder, as evidenced by the reduction, arrest, or elimination of any clinical or diagnostic symptoms, by administering an immunomodulatory protein, or an engineered cell of the invention, alone or in combination with another compound described herein. "Treating," "treatment," or "therapy" also means reducing the severity of symptoms in an acute or chronic disease or disorder, or reducing the relapse rate, as in the case of a relapsing or remitting autoimmune disease course, or reducing inflammation in the case of the inflammatory aspect of an autoimmune disease. "Preventing" a disease or disorder, "prophylaxis," or "prevention" as used in the context of the present invention refers to administering an immunomodulatory protein of the invention, or an engineered cell expressing an immunomodulatory protein, alone or in combination with another compound, to prevent the occurrence or development of the disease or disorder, or some or all of the symptoms of the disease or disorder, or to reduce the likelihood of developing the disease or disorder. For example, in the context of cancer, the terms "treating" cancer or "inhibiting" cancer, "inhibiting" or "inhibition" of cancer refer, without limitation, to at least one of a statistically significant decrease in tumor growth rate, cessation of tumor growth, or a decrease in tumor size, mass, metabolic activity, or volume, as measured by standard criteria such as Response Evaluation Criteria for Solid Tumors (RECIST), or a statistically significant increase in progression-free survival (PFS) or overall survival (OS).

[0314] VI. Illustrative Embodiments Among the aspects provided are the following: 1. A pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal or oligoclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, the population comprising at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 2.0% within the population, and at least 90% of the cells in the composition are CD3+ T cells. 2. The pharmaceutical composition of embodiment 1, wherein at least 11 different TCR clonotypes have a frequency of at least 2.0% within said population. 3. The pharmaceutical composition of embodiment 1, wherein at least 12 different TCR clonotypes have a frequency of at least 2.0% within said population. 4. A pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal or oligoclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, the population comprising at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 1.0% within the population, and at least 90% of the cells in the composition are CD3+ T cells. 5. The pharmaceutical composition of embodiment 4, wherein at least 11 different TCR clonotypes have a frequency of at least 1.0% in said population. 6. The pharmaceutical composition of embodiment 4, wherein at least 12 different TCR clonotypes have a frequency of at least 1.0% in said population. 7. The pharmaceutical composition of any one of aspects 1-6, wherein between 8 and 15 different T cell receptor (TCR) clonotypes comprise at least 50% of the TCR frequency within said population. 8. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 8 to 15 different T cell receptor (TCR) clonotypes constitute at least 50% of the TCR frequency within the population. 9. The pharmaceutical composition of any one of aspects 1-8, wherein 9 to 12 different TCR clonotypes constitute at least 50% of the TCR frequency within said population. 10. The pharmaceutical composition of any one of aspects 1-9, wherein said TCR clonotype exhibits reactivity to at least one CD4 antigen and at least one CD8 antigen. 11. The pharmaceutical composition of any one of aspects 1 to 10, wherein the TCR clonotype exhibits reactivity to between 2 and 100 different peptide antigens. 12. The pharmaceutical composition of any one of aspects 1-11, wherein the TCR clonotype exhibits reactivity to 10 to 40 different peptide antigens. 13. The pharmaceutical composition of any one of aspects 1-11, wherein the TCR clonotypes exhibit reactivity to 2 to 6 different peptide antigens. 14. The pharmaceutical composition of any one of aspects 1 to 11, wherein the TCR clonotype exhibits reactivity to two to four types of peptide antigens. 15. The pharmaceutical composition of any one of aspects 1 to 11, wherein the TCR clonotype exhibits reactivity to two different peptide antigens. 16. The pharmaceutical composition of any one of aspects 1-11 and 13-15, wherein the TCR clonotype exhibits reactivity to one CD8 antigen and one CD4 antigen. 17. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 10 to 100 different T cell receptor (TCR) clonotypes are present within the population. 18. The pharmaceutical composition of aspect 17, wherein the TCR clonotype exhibits reactivity to 10 to 40 different peptide antigens. 19. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 20 to 100 different T cell receptor (TCR) clonotypes are present within the population. 20. The pharmaceutical composition of embodiment 19, wherein between 20 and 60 different TCR clonotypes are present in said population. 21. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein the top 40 TCR clonotypes constitute at least 75% of the TCR frequency within the population. 22. The pharmaceutical composition of any one of aspects 5-21, wherein at least 90% of the cells in the population are CD3+ T cells. 23. The pharmaceutical composition of any one of aspects 1-22, wherein the TCR clonotype exhibits reactivity to at least one CD8 antigen and at least one CD4 antigen. 24. The pharmaceutical composition of any one of aspects 1-23, wherein at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity. 25. A pharmaceutical T lymphocyte infiltrating (TIL) composition enriched in tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, at least 90% of the cells in the composition are CD3+ T cells, and at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity. 26. The pharmaceutical composition of embodiment 24 or embodiment 25, wherein at least 25% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition are neoantigen-reactive. 27. The pharmaceutical composition of embodiment 24 or embodiment 25, wherein at least 30% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition are neoantigen-reactive. 28. The pharmaceutical composition of embodiment 24 or embodiment 25, wherein at least 40% of the CD8+ T cells and / or at least 30% of the CD4+ T cells in the composition are neoantigen-reactive. 29. The pharmaceutical composition of any one of aspects 19 to 28, wherein neoantigen reactivity is determined in a co-culture assay with peptide-loaded autologous APCs (e.g., as described in Example 2) by one or more of upregulation of CD134 and CD137, IFN-γ production, TNF-α production, granzyme B production, or degranulation, and optionally degranulation is determined based on CD107 expression. 30. The pharmaceutical composition of any one of aspects 1-29, wherein the TIL composition is characterized by at least a 1.5-fold increased percentage of cells positive for CD134 and CD137 compared to a bulk TIL population in a co-culture assay with peptide-loaded autologous APCs, and optionally by at least a 2-fold, at least a 3-fold, or at least a 4-fold increase in CD134-positive cells and CD137-positive cells compared to a bulk TIL population. 31. The pharmaceutical composition of any one of aspects 1-11, wherein in a co-culture assay with peptide-loaded autologous APCs, more than 30% of the cells in the TIL composition are positive for CD134 and CD137, and optionally, more than about 35%, more than about 40%, or more than about 45% of the cells are positive for CD134 and CD137. 32. The pharmaceutical composition of any one of aspects 1-31, wherein in a co-culture assay with peptide-loaded autologous APCs, greater than 48% of the cells in the TIL composition are positive for CD134 and CD137, and optionally greater than about 50%, greater than about 60%, or greater than about 70% of the cells are positive for CD134 and CD137. 33. The pharmaceutical composition of any one of aspects 1 to 32, wherein the TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ production greater than 1,000 pg / mL; (ii) TNF-α production greater than 100 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 10,000 pg / mL. 34. The pharmaceutical composition of any one of aspects 1 to 33, wherein the TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ production greater than 100,000 pg / mL; (ii) TNF-α production greater than 250 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 50,000 pg / mL. 35. A pharmaceutical T lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ production greater than 1,000 pg / mL; (ii) TNF-α production greater than 100 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 10,000 pg / mL. 36. A pharmaceutical T lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ production greater than 100,000 pg / mL; (ii) TNF-α production greater than 250 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 50,000 pg / mL. 37. The pharmaceutical composition of any one of aspects 33-36, wherein the TIL composition is characterized by at least two of criteria (i)-(iv). 38. The pharmaceutical composition of any one of aspects 33-36, wherein the TIL composition is characterized by at least three of criteria (i)-(iv). 39. The pharmaceutical composition of any one of aspects 33 to 36, wherein the TIL composition is characterized by criteria (i) to (iv). 40. The pharmaceutical composition of any one of aspects 29-39, wherein the TIL composition is characterized by IFN-γ production of greater than 2,500 pg / mL, greater than 5,000 pg / mL, greater than 10,000 pg / mL, greater than 25,000 pg / mL, greater than 50,000 pg / mL, greater than 100,000 pg / mL, greater than 200,000 pg / mL, greater than 250,000 pg / mL, greater than 500,000 pg / mL, or greater than 1,000,000 pg / mL. 41. The pharmaceutical composition of any one of aspects 29-40, wherein the TIL composition is characterized by IFN-γ production of greater than 250,000 pg / mL, greater than 500,000 pg / mL, or greater than 1,000,000 pg / mL. 42. The pharmaceutical composition of any one of aspects 29-41, wherein the TIL composition is characterized by TNF-α production of greater than 200 pg / mL, greater than 500 pg / mL, greater than 1000 pg / mL, or greater than 2000 pg / mL. 43. The pharmaceutical composition of any one of aspects 29-42, wherein the TIL composition is characterized by TNF-α production of greater than 500 pg / mL, greater than 1000 pg / mL, or greater than 2000 pg / mL. 44. The pharmaceutical composition of any one of aspects 29-43, wherein the TIL composition is characterized by greater than 15% CD107a+ cells, greater than 20% CD107a cells, or greater than 25% CD107a+ cells. 45. The pharmaceutical composition of any one of aspects 29-44, wherein the TIL composition is characterized by Granzyme B production of greater than 15,000 pg / mL, greater than 25,000 pg / mL, greater than 50,000 pg / mL, greater than 100,000 pg / mL, greater than 200,000 pg / mL, greater than 300,000 pg / mL, greater than 400,000 pg / mL, or greater than 500,000 pg / mL. 46. ​​The pharmaceutical composition of any one of aspects 29-45, wherein the TIL composition is characterized by Granzyme B production of greater than 200,000 pg / mL, greater than 300,000 pg / mL, greater than 400,000 pg / mL, or greater than 500,000 pg / mL. 47. The pharmaceutical composition of any one of aspects 1 to 46, wherein the TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ that is 50-fold greater than bulk TIL compositions not enriched for tumor-reactive T cells; (ii) TNF-α that is 300-fold or greater than bulk TIL composition that is not enriched for tumor-reactive T cells; or (iii) Granzyme B that is 15-fold greater than bulk TIL composition not enriched for tumor-reactive T cells. 48. A pharmaceutical T lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ that is 50-fold greater than bulk TIL compositions not enriched for tumor-reactive T cells; (ii) TNF-α that is 300-fold or greater than bulk TIL composition that is not enriched for tumor-reactive T cells; or (iii) Granzyme B that is 15-fold greater than bulk TIL composition not enriched for tumor-reactive T cells. 49. The pharmaceutical composition of embodiment 47 or embodiment 48, wherein said TIL composition is characterized by criteria (i) and (ii). 50. The pharmaceutical composition of embodiment 47 or embodiment 48, wherein said TIL composition is characterized by criteria (i) and (iii). 51. The pharmaceutical composition of embodiment 47 or embodiment 48, wherein said TIL composition is characterized by criteria (ii) and (iii). 52. The pharmaceutical composition of embodiment 47 or embodiment 48, wherein said TIL composition is characterized by criteria (i), (ii), and (iii). 53. The pharmaceutical composition of any one of aspects 1-52, characterized by a greater number of CD4+ T cells than CD8+ T cells. 54. The pharmaceutical composition of any one of aspects 1-52, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 1:5. 55. The pharmaceutical composition of any one of aspects 1-52, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:1. 56. The pharmaceutical composition of any one of aspects 1-55, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 10:1 to 25:1, or about 10:1 to 25:1, optionally 20:1 or about 20:1. 57. The pharmaceutical composition of any one of aspects 1-56, wherein greater than 50% of said CD3+ T cells, and optionally greater than 50% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype. 58. The pharmaceutical composition of any one of aspects 1-57, wherein greater than 75% of said CD3+ T cells, and optionally greater than 75% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype. 59. The pharmaceutical composition of any one of aspects 1-58, wherein greater than 80% of said CD3+ T cells, and optionally greater than 80% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype. 60. The pharmaceutical composition of any one of aspects 1-58, wherein greater than 85% of said CD3+ T cells, and optionally greater than 85% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype. 61. The pharmaceutical composition of any one of aspects 1-58, wherein greater than 90% of said CD3+ T cells, and optionally greater than 90% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype. 62. The effector memory phenotype is CD45RA - , CD45RO+, CD62L - 62. The pharmaceutical composition of any one of embodiments 58-61, characterized by surface marker expression that is one or more of CCR7-, CD28- and CD27-. 63. The effector memory phenotype is characterized by the surface marker expression CD45RA - , CD45RO+, CD62L - , and CCR7 - 63. The pharmaceutical composition of any one of embodiments 58 to 62, wherein 64. The effector memory phenotype is characterized by the surface marker expression CD45RA - , CD45RO + , CD62L - , CCR7 - , CD28 - and CD27 - 63. The pharmaceutical composition of any one of embodiments 58 to 62, wherein 65. The effector memory phenotype is characterized by the surface marker expression CD45RA - and CCR7 -- 63. The pharmaceutical composition of any one of embodiments 58 to 62, wherein 66. More than 95% of the CD4+ T cells and CD8+ T cells in the composition express PD-1 - 66. The pharmaceutical composition of any one of embodiments 1-65, wherein 67. More than 80% of the CD4+ T cells and CD8+ T cells in the composition are LAG3 - 67. The pharmaceutical composition of any one of embodiments 1-66, wherein 68. The number of cells in the composition, or the number of viable cells thereof, is at least 2 x 10 7 68. The pharmaceutical composition of any one of embodiments 1 to 67, wherein 69. The number of cells in the composition, or the number of viable cells thereof, is 2 x 10 7 pieces~20×10 9 pieces, 2×10 7 pieces~10×10 9 pieces, 2×10 7 pieces~2×10 9 pieces, 2×10 7 pieces~2×10 8 pieces, 2×10 8 pieces~20×10 9 pieces, 2×10 8 pieces~10×10 9 pieces, 2×10 8 pieces~2×109 pieces, 2×10 9 pieces~20×10 9 pieces, 2×10 9 pieces~10×10 9 pieces, or 10 x 10 9 pieces~20×10 9 pieces, or approximately 2 x 10 7 pieces~20×10 9 pieces, approximately 2×10 7 pieces~10×10 9 pieces, approximately 2×10 7 pieces~2×10 9 pieces, approximately 2×10 7 pieces~2×10 8 pieces, approximately 2×10 8 pieces~20×10 9 pieces, approximately 2×10 8 pieces~10×10 9 pieces, approximately 2×10 8 pieces~2×10 9 pieces, approximately 2×10 9 pieces~20×10 9 pieces, approximately 2×10 9 pieces~10×10 9 pieces, or about 10 x 10 9 pieces~20×10 9 69. The pharmaceutical composition of any one of embodiments 1-68, wherein (inclusive) 70. The pharmaceutical composition of any one of aspects 1-69, for treating a tumor in a patient. 71. The pharmaceutical composition of any one of aspects 1-70, wherein the tumor is a colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor. 72. The pharmaceutical composition of any one of aspects 1-71, wherein the tumor is derived from a human subject. 73. The pharmaceutical composition of embodiment 72, which is for autologous adoptive therapy for the human subject. 74. The pharmaceutical composition of any one of aspects 1-73, comprising a pharmaceutically acceptable excipient. 75. The pharmaceutical composition of any one of aspects 1-74, comprising a cryoprotectant. 76. The pharmaceutical composition of any one of aspects 1-75, which is a liquid composition. 77. The pharmaceutical composition of embodiment 76, which has been frozen and thawed. 78. The pharmaceutical composition of any one of aspects 1-77, wherein the volume of the composition is 1 mL to 500 mL. 79. The pharmaceutical composition of any one of aspects 1-78, which is frozen. 80. A pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched in tumor-reactive T cells for treating a tumor in a patient, the pharmaceutical composition comprising a multiclonal or oligoclonal population of T cells comprising CD4+ T cells and CD8+ T cells derived from the pa...

Claims

1. A pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, the population comprising at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 2.0% within the population, and at least 90% of the cells in the composition being CD3+ T cells.

2. 2. The pharmaceutical composition of claim 1, wherein at least 11 different TCR clonotypes have a frequency of at least 2.0% in the population.

3. 2. The pharmaceutical composition of claim 1, wherein at least 12 different TCR clonotypes have a frequency of at least 2.0% in the population.

4. A pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of T cells comprising tumor-derived CD4+ T cells and CD8+ T cells, the population comprising at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 1.0% within the population, and at least 90% of the cells in the composition being CD3+ T cells.

5. 5. The pharmaceutical composition of claim 4, wherein at least 11 different TCR clonotypes have a frequency of at least 1.0% in the population.

6. 5. The pharmaceutical composition of claim 4, wherein at least 12 different TCR clonotypes have a frequency of at least 1.0% in the population.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein 8 to 15 different T cell receptor (TCR) clonotypes comprise at least 50% of the TCR frequency in said population.

8. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 8 to 15 different T cell receptor (TCR) clonotypes constitute at least 50% of the TCR frequency within the population.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein 9 to 12 different TCR clonotypes constitute at least 50% of the TCR frequency in said population.

10. 10. The pharmaceutical composition of any one of claims 1 to 9, wherein said TCR clonotype exhibits reactivity to at least one CD4 antigen and at least one CD8 antigen.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the TCR clonotype exhibits reactivity to 2 to 100 different peptide antigens.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the TCR clonotype exhibits reactivity to 10 to 40 different peptide antigens.

13. The pharmaceutical composition of any one of claims 1 to 11, wherein the TCR clonotype exhibits reactivity to 2 to 6 different peptide antigens.

14. The pharmaceutical composition of any one of claims 1 to 11, wherein the TCR clonotype exhibits reactivity to two to four types of peptide antigens.

15. The pharmaceutical composition of any one of claims 1 to 11, wherein the TCR clonotype exhibits reactivity to two types of peptide antigens.

16. 16. The pharmaceutical composition of any one of claims 1 to 11 and 13 to 15, wherein said TCR clonotype exhibits reactivity to one CD8 antigen and one CD4 antigen.

17. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 10 to 100 different T cell receptor (TCR) clonotypes are present within the population.

18. 18. The pharmaceutical composition of claim 17, wherein the TCR clonotype exhibits reactivity to 10 to 40 different peptide antigens.

19. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein 20 to 100 different T cell receptor (TCR) clonotypes are present within the population.

20. 20. The pharmaceutical composition of claim 19, wherein between 20 and 60 different TCR clonotypes are present in said population.

21. A pharmaceutical composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising a multiclonal population of tumor-infiltrating lymphocytes comprising tumor-derived CD4+ T cells and CD8+ T cells, wherein the top 40 TCR clonotypes constitute at least 75% of the TCR frequency within the population.

22. 22. The pharmaceutical composition of any one of claims 5 to 21, wherein at least 90% of the cells in the population are CD3+ T cells.

23. 23. The pharmaceutical composition of any one of claims 1 to 22, wherein said TCR clonotype exhibits reactivity to at least one CD8 antigen and at least one CD4 antigen.

24. 24. The pharmaceutical composition of any one of claims 1 to 23, wherein at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

25. A pharmaceutical T-lymphocyte infiltration (TIL) composition enriched in tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, at least 90% of the cells in the composition are CD3+ T cells, and at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

26. 26. The pharmaceutical composition of claim 24 or claim 25, wherein at least 25% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

27. 26. The pharmaceutical composition of claim 24 or claim 25, wherein at least 30% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

28. 26. The pharmaceutical composition of claim 24 or claim 25, wherein at least 40% of the CD8+ T cells and / or at least 30% of the CD4+ T cells in the composition exhibit neoantigen reactivity.

29. The pharmaceutical composition of any one of claims 19 to 28, wherein neoantigen reactivity is determined in a co-culture assay with peptide-loaded autologous APCs (e.g., as described in Example 2) by one or more of upregulation of CD134 and CD137, IFN-γ production, TNF-α production, granzyme B production or degranulation, and optionally degranulation is determined based on CD107 expression.

30. 30. The pharmaceutical composition of any one of claims 1 to 29, wherein the TIL composition is characterized by at least a 1.5-fold increased percentage of cells positive for CD134 and CD137 compared to a bulk TIL population in a co-culture assay with peptide-loaded autologous APCs, and optionally by at least a 2-fold, at least a 3-fold, or at least a 4-fold increase in CD134-positive cells and CD137-positive cells compared to a bulk TIL population.

31. The pharmaceutical composition of any one of claims 1 to 11, wherein in a co-culture assay with peptide-loaded autologous APCs, more than 30% of the cells in the TIL composition are positive for CD134 and CD137, and optionally more than about 35%, more than about 40%, or more than about 45% of the cells are positive for CD134 and CD137.

32. 32. The pharmaceutical composition of any one of claims 1 to 31, wherein in a co-culture assay with peptide-loaded autologous APCs, more than 48% of the cells in the TIL composition are positive for CD134 and CD137, and optionally, more than about 50%, more than about 60%, or more than about 70% of the cells are positive for CD134 and CD137.

33. The pharmaceutical composition of any one of claims 1 to 32, wherein said TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ production greater than 1,000 pg / mL; (ii) TNF-α production greater than 100 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 10,000 pg / mL.

34. The pharmaceutical composition of any one of claims 1 to 33, wherein said TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ production greater than 100,000 pg / mL; (ii) TNF-α production greater than 250 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 50,000 pg / mL.

35. 1. A pharmaceutical T-lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized in an in vitro co-culture assay with peptide-loaded autologous APCs by at least one of the following criteria: (i) IFN-γ production greater than 1,000 pg / mL; (ii) TNF-α production greater than 100 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 10,000 pg / mL.

36. 1. A pharmaceutical T-lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized in an in vitro co-culture assay with peptide-loaded autologous APCs by at least one of the following criteria: (i) IFN-γ production greater than 100,000 pg / mL; (ii) TNF-α production greater than 250 pg / mL; (iii) greater than 10% CD107a+ cells; and (iv) Granzyme B production greater than 50,000 pg / mL.

37. The pharmaceutical composition of any one of claims 33 to 36, wherein said TIL composition is characterized by at least two of criteria (i) to (iv).

38. The pharmaceutical composition of any one of claims 33 to 36, wherein said TIL composition is characterized by at least three of criteria (i) to (iv).

39. The pharmaceutical composition of any one of claims 33 to 36, wherein said TIL composition is characterized by criteria (i) to (iv).

40. 40. The pharmaceutical composition of any one of claims 29 to 39, wherein the TIL composition is characterized by IFN-γ production of greater than 2,500 pg / mL, greater than 5,000 pg / mL, greater than 10,000 pg / mL, greater than 25,000 pg / mL, greater than 50,000 pg / mL, greater than 100,000 pg / mL, greater than 200,000 pg / mL, greater than 250,000 pg / mL, greater than 500,000 pg / mL, or greater than 1,000,000 pg / mL.

41. 41. The pharmaceutical composition of any one of claims 29 to 40, wherein the TIL composition is characterized by IFN-γ production of greater than 250,000 pg / mL, greater than 500,000 pg / mL, or greater than 1,000,000 pg / mL.

42. 42. The pharmaceutical composition of any one of claims 29 to 41, wherein the TIL composition is characterized by TNF-α production of greater than 200 pg / mL, greater than 500 pg / mL, greater than 1000 pg / mL, or greater than 2000 pg / mL.

43. 43. The pharmaceutical composition of any one of claims 29 to 42, wherein the TIL composition is characterized by TNF-α production of greater than 500 pg / mL, greater than 1000 pg / mL, or greater than 2000 pg / mL.

44. The pharmaceutical composition of any one of claims 29 to 43, wherein the TIL composition is characterized by more than 15% CD107a+ cells, more than 20% CD107a+ cells, or more than 25% CD107a+ cells.

45. 45. The pharmaceutical composition of any one of claims 29 to 44, wherein the TIL composition is characterized by granzyme B production of greater than 15,000 pg / mL, greater than 25,000 pg / mL, greater than 50,000 pg / mL, greater than 100,000 pg / mL, greater than 200,000 pg / mL, greater than 300,000 pg / mL, greater than 400,000 pg / mL, or greater than 500,000 pg / mL.

46. 46. ​​The pharmaceutical composition of any one of claims 29 to 45, wherein the TIL composition is characterized by granzyme B production of greater than 200,000 pg / mL, greater than 300,000 pg / mL, greater than 400,000 pg / mL, or greater than 500,000 pg / mL.

47. The pharmaceutical composition of any one of claims 1 to 46, wherein said TIL composition is characterized by at least one of the following criteria in an in vitro co-culture assay with peptide-loaded autologous APCs: (i) IFN-γ that is 50-fold greater than bulk TIL compositions not enriched for tumor-reactive T cells; (ii) TNF-α that is 300-fold or greater than that of a bulk TIL composition that is not enriched for tumor-reactive T cells; or (iii) Granzyme B that is 15-fold greater than bulk TIL composition not enriched for tumor-reactive T cells.

48. 1. A pharmaceutical T-lymphocyte infiltrating (TIL) composition enriched for tumor-reactive T cells, the pharmaceutical composition comprising tumor-infiltrating lymphocytes including tumor-derived CD4+ T cells and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized in an in vitro co-culture assay with peptide-loaded autologous APCs by at least one of the following criteria: (i) IFN-γ that is 50-fold greater than bulk TIL compositions not enriched for tumor-reactive T cells; (ii) TNF-α that is 300-fold or greater than that of a bulk TIL composition that is not enriched for tumor-reactive T cells; or (iii) Granzyme B that is 15-fold greater than bulk TIL composition not enriched for tumor-reactive T cells.

49. 49. The pharmaceutical composition of claim 47 or claim 48, wherein the TIL composition is characterized by criteria (i) and (ii).

50. 49. The pharmaceutical composition of claim 47 or claim 48, wherein the TIL composition is characterized by criteria (i) and (iii).

51. 49. The pharmaceutical composition of claim 47 or claim 48, wherein the TIL composition is characterized by criteria (ii) and (iii).

52. 49. The pharmaceutical composition of claim 47 or claim 48, wherein the TIL composition is characterized by criteria (i), (ii) and (iii).

53. 53. The pharmaceutical composition of any one of claims 1 to 52, characterized by a greater number of CD4+ T cells than CD8+ T cells.

54. 53. The pharmaceutical composition of any one of claims 1 to 52, wherein the ratio of CD4+ T cells to CD8+ T cells in said composition is 5:1 to 1:

5.

55. 53. The pharmaceutical composition of any one of claims 1-52, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:

1.

56. 56. The pharmaceutical composition of any one of claims 1-55, wherein the ratio of CD4+ T cells to CD8+ T cells in said composition is between 10:1 and 25:1, or about 10:1 and 25:1, optionally 20:1 or about 20:

1.

57. 57. The pharmaceutical composition of any one of claims 1-56, wherein greater than 50% of said CD3+ T cells, and optionally greater than 50% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype.

58. 58. The pharmaceutical composition of any one of claims 1-57, wherein greater than 75% of said CD3+ T cells, and optionally greater than 75% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype.

59. 59. The pharmaceutical composition of any one of claims 1-58, wherein greater than 80% of said CD3+ T cells, and optionally greater than 80% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype.

60. 59. The pharmaceutical composition of any one of claims 1-58, wherein greater than 85% of said CD3+ T cells, and optionally greater than 85% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype.

61. 59. The pharmaceutical composition of any one of claims 1-58, wherein greater than 90% of said CD3+ T cells, and optionally greater than 90% of said CD4+ T cells and CD8+ T cells, express markers of an effector memory phenotype.

62. The effector memory phenotype is - , CD45RO+, CD62L - 62. The pharmaceutical composition of any one of claims 58 to 61, characterized by surface marker expression that is one or more of: CCR7-, CD28- and CD27-.

63. The effector memory phenotype is characterized by the expression of the surface marker CD45RA - , CD45RO+, CD62L - , and CCR7 - 63. The pharmaceutical composition according to any one of claims 58 to 62, characterized in that

64. The effector memory phenotype is characterized by the expression of the surface marker CD45RA - , CD45RO ＋ , CD62L - , CCR7 - , CD28 - and CD27 - 63. The pharmaceutical composition according to any one of claims 58 to 62, characterized in that

65. The effector memory phenotype is characterized by the expression of the surface marker CD45RA - and CCR7 -- 63. The pharmaceutical composition according to any one of claims 58 to 62, characterized in that

66. More than 95% of the CD4+ T cells and CD8+ T cells in the composition are PD-1 - 66. The pharmaceutical composition of any one of claims 1 to 65, wherein

67. greater than 80% of the CD4+ T cells and CD8+ T cells in the composition are LAG3 - 67. The pharmaceutical composition of any one of claims 1 to 66, wherein

68. The number of cells in the composition, or the number of viable cells thereof, is at least 2×10 7 68. The pharmaceutical composition of any one of claims 1 to 67,

69. The number of cells in the composition, or the number of viable cells thereof, is 2×10 7 pieces ~ 20×10 9 pieces, 2×10 7 pieces ~ 10×10 9 pieces, 2×10 7 pieces ~ 2×10 9 pieces, 2×10 7 pieces ~ 2×10 8 pieces, 2×10 8 pieces ~ 20×10 9 pieces, 2×10 8 pieces ~ 10×10 9 pieces, 2×10 8 pieces ~ 2×10 9 pieces, 2×10 9 pieces ~ 20×10 9 pieces, 2×10 9 pieces ~ 10×10 9 pieces, or 10 x 10 9 pieces ~ 20×10 9 pieces, or approximately 2 x 10 7 pieces ~ 20×10 9 pieces, approximately 2×10 7 pieces ~ 10×10 9 pieces, approximately 2×10 7 pieces ~ 2×10 9 pieces, approximately 2×10 7 pieces ~ 2×10 8 pieces, approximately 2×10 8 pieces ~ 20×10 9 pieces, approximately 2×10 8 pieces ~ 10×10 9 pieces, approximately 2×10 8 pieces ~ 2×10 9 pieces, approximately 2×10 9 pieces ~ 20×10 9 pieces, approximately 2×10 9 pieces ~ 10×10 9 pieces, or about 10 x 10 9 pieces ~ 20×10 9 69. The pharmaceutical composition of any one of claims 1 to 68, wherein (inclusive)

70. 70. The pharmaceutical composition of any one of claims 1 to 69, for treating a tumor in a patient.

71. 71. The pharmaceutical composition of any one of claims 1 to 70, wherein the tumor is a colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor.

72. 72. The pharmaceutical composition of any one of claims 1 to 71, wherein the tumor is derived from a human subject.

73. 73. The pharmaceutical composition of claim 72, for autologous adoptive therapy for the human subject.

74. 74. The pharmaceutical composition of any one of claims 1 to 73, comprising a pharmaceutically acceptable excipient.

75. 75. The pharmaceutical composition of any one of claims 1 to 74, comprising a cryoprotectant.

76. 76. The pharmaceutical composition of any one of claims 1 to 75, which is a liquid composition.

77. 77. The pharmaceutical composition of claim 76, which has been frozen and thawed.

78. 78. The pharmaceutical composition of any one of claims 1-77, wherein the volume of the composition is between 1 mL and 500 mL.

79. 79. The pharmaceutical composition of any one of claims 1-78, which is frozen.

80. 1. A pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched in tumor-reactive T cells for treating a tumor in a patient, the pharmaceutical composition comprising a multiclonal population of T cells comprising CD4+ T cells and CD8+ T cells derived from the patient's tumor, the population comprising at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 2.0% within the population, and at least 90% of the cells in the composition being CD3+ T cells.

81. 1. A pharmaceutical T-lymphocyte infiltrate (TIL) composition enriched in tumor-reactive T cells for treating a tumor in a patient, the pharmaceutical composition comprising a multiclonal population of T cells comprising CD4+ T cells and CD8+ T cells derived from the patient's tumor, the population comprising at least 10 different T-cell receptor (TCR) clonotypes, each having a frequency of at least 1.0% within the population, and at least 90% of the cells in the composition being CD3+ T cells.

82. The pharmaceutical composition of any one of claims 1 to 81, wherein the TIL composition is produced by an ex vivo method comprising expanding tumor-reactive T cells from a donor subject, and the tumor-reactive T cells are co-cultured with autologous antigen-presenting cells and peptide neoantigens.

83. The pharmaceutical composition of any one of claims 1 to 82, wherein said TIL composition is produced by a method comprising the steps of: a. providing tumor cells dissociated from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first T cell population comprising CD4+ T cells and CD8+ T cells; b. culturing the first population of T cells with recombinant IL-2 added at a concentration of 3000 IU / mL to 6000 IU / mL, inclusive, for 14 to 28 days to generate a second population of T cells; c. co-culturing the second T cell population with autologous antigen-presenting cells (APCs) for 12 to 48 hours with recombinant IL-2 added at a concentration of 100 IU / mL to 1000 IU / mL to generate a third T cell population, wherein the APCs are loaded with a pool of peptide neoantigens derived from the tumor, each peptide being 13 to 40 amino acids in length and loaded at a concentration of 100 ng / mL per peptide, at a ratio of the second T cell population to APCs of 2:1 to 10:1; d. selecting cells from the third population of T cells that are surface positive for CD134 and / or CD137 to generate a fourth population of T cells; and e. Expanding tumor infiltrating lymphocytes (TILs) by incubating the fourth T cell population with irradiated human peripheral blood mononuclear cells (iPBMCs) at a ratio of 100 to 500 iPBMCs to cells of the fourth T cell population for 12 to 16 days with recombinant IL-2 added at a concentration of 3000 IU / mL to 6000 IU / mL (inclusive) and 10 to 50 ng / mL of anti-CD3 antibody (OKT3) to produce a therapeutic composition of tumor-reactive cells-enriched tumor infiltrating lymphocytes (TILs).

84. 84. A frozen composition comprising the pharmaceutical composition of any one of claims 1 to 83 and a cryoprotectant.

85. A method for producing a T lymphocyte infiltrate (TIL) composition enriched for tumor-reactive T cells, comprising the steps of: a. providing tumor cells dissociated from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first T cell population comprising CD4+ T cells and CD8+ T cells; b. culturing the first population of T cells with recombinant IL-2 added at a concentration of about 3000 IU / mL to 6000 IU / mL, inclusive, for about 14 to 28 days to generate a second population of T cells; c. co-culturing the second T cell population with autologous antigen-presenting cells (APCs) for about 12 to 48 hours with recombinant IL-2 added at a concentration of 100 IU / mL to 1000 IU / mL to generate a third T cell population, wherein the APCs are loaded with a pool of peptide neoantigens derived from the tumor, each peptide being 13 to 40 amino acids in length and loaded at a concentration of 100 ng / mL per peptide, and the ratio of the second T cell population to APCs is about 2:1 to 10:1; d. selecting cells from the third population of T cells that are surface positive for CD134 and / or CD137 to generate a fourth population of T cells; and e. Expanding tumor infiltrating lymphocytes (TILs) by incubating the fourth T cell population with irradiated human peripheral blood mononuclear cells (iPBMCs) at a ratio of about 100 to 500 iPBMCs to cells of the fourth T cell population for 12 to 16 days with recombinant IL-2 added at a concentration of about 3000 IU / mL to 6000 IU / mL, inclusive, and 10 to 50 ng / mL of anti-CD3 antibody (OKT3) to produce a therapeutic composition of tumor-reactive cells-enriched tumor infiltrating lymphocytes (TILs).

86. 85. A method of treating a subject with cancer, comprising administering to said subject a therapeutic dose of the composition of any one of claims 1-84.

87. The therapeutically effective dose is about 1×10 9 ~10×10 9 87. The method of claim 86, wherein the T cells are T cells.

88. 87. The method of claim 86, wherein said therapeutically effective dose is greater than 1 million to less than 100 million T cells per kilogram of body weight.

89. 87. The method of claim 86, wherein said therapeutically effective dose is greater than 1 million to less than 10 million T cells per kilogram of body weight.

90. 87. The method of claim 86, wherein said therapeutically effective dose is at or about 10 million to at or about 50 million T cells per kilogram of body weight.

91. 91. The method of any one of claims 86-90, wherein said cells of said therapeutic composition are autologous to said subject.

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