Compositions and methods for generating neo-antigen reactive tumor infiltrating lymphocytes

A pharmaceutical T lymphocyte infiltrating (TIL) composition enriched in tumor reactive T cells, the pharmaceutical composition comprising tumor infiltrating lymphocytes comprising CD4+ and CD8+ T cells from a tumor, wherein at least 90% of cells in the composition are CD3+ T cells and 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 APC:

• i) IFN-γ production that is greater than 1,000 pg/mL;
• (ii) TNF-alpha production that is greater than 100 pg/ml;
• (iii) greater than 10% CD107a+ cells;
• and
• (iv) granzyme B production that is greater than 10,000 pg/mL.

A pharmaceutical T lymphocyte infiltrating (TIL) composition enriched in tumor reactive T cells, the pharmaceutical composition comprising tumor infiltrating lymphocytes comprising CD4+ and CD8+ T cells from a tumor, wherein at least 90% of cells in the composition are CD3+ T cells and 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 APC:

• i) IFN-γ production that is greater than 100,000 pg/mL;
• (ii) TNF-alpha production that is greater than 250 pg/mL;
• (iii) greater than 10% CD107a+ cells;
• and
• (iv) granzyme B production that is greater than 50,000 pg/mL.

A pharmaceutical T lymphocyte infiltrating (TIL) composition enriched in tumor reactive T cells, the pharmaceutical composition comprising tumor infiltrating lymphocytes comprising CD4+ and CD8+ T cells from a tumor, wherein at least 90% of cells in the composition are CD3+ T cells and 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 APC:

• i) IFN-γ that is 50-fold or higher than a bulk TIL composition that is not enriched for tumor reactive T cells;
• ii) TNF-α that is 300-fold or higher than a bulk TIL composition that is not enriched for tumor reactive T cells;
• or
• iii) granzyme B that is 15-fold or higher from a bulk TIL composition that is not enriched for tumor reactive T cells.

A method of producing a T lymphocyte infiltrating (TIL) composition enriched in tumor reactive T cells, the method comprising:

• a. providing dissociated tumor cells from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first population of T cells that comprise CD4+ and CD8+ T cells;
• b. culturing the first population of T cells with recombinant IL-2 added at a concentration between about 3000 IU/mL and 6000 IU/mL, inclusive, for about 14 to 28 days to produce a second population of T cells;
• c. co-culturing the second population of T cells for about 12 to 48 hours with autologous antigen presenting cells (APCs) with recombinant IL-2 added at a concentration of 100 IU/mL to 1000 IU/mL to produce a third population of T cells, wherein the APCs are loaded with a pool of peptide neoantigens from the tumor, wherein each peptide is 13-40 amino acids in length and is loaded at a concentration of 100 ng/ml per peptide, and wherein the ratio of the second population of T cells 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 produce a fourth population of T cells;
• and
• e. expanding tumor infiltrating lymphocytes (TILs) by incubating the fourth population of T cells with irradiated human peripheral blood mononuclear cells (iPBMCs) at a ratio of about 100 to 500 iPBMC to cells of the fourth population of T cells with recombinant IL-2 added at a concentration between about 3000 IU/mL and 6000 IU/mL, inclusive, and 10 to 50 ng/ml anti-CD3 antibody (OKT3) for 12 to 16 days to produce a therapeutic composition of TILs enriched in tumor reactive cells.