Processing of tumor infiltrating lymphocytes

The method of any preceding claim, wherein the first cell culture medium comprises fetal bovine serum (FBS) and about 2000 IU/mL of IL-2,

• wherein step (b) comprises initially seeding the processed resected tumor product in about 20 to about 40 mL of the cell culture medium in a 70-mL cell culture bag,
• wherein step (b) comprises initially seeding the processed resected tumor product in a whole culture volume of cell culture medium, adding the cell culture medium at a first time point, conditionally adding the cell culture medium at a second time point based on cell concentration, and optionally conditionally adding the cell culture medium at a third time point based on cell concentration,
• wherein a half culture volume (0.5×) of cell culture medium is added at the first time point,
• wherein adding the cell culture medium at the second and third time points is determined based on CD45+ viable cell density,
• wherein a half culture volume (0.5×) of cell culture medium is added at the second time point if CD45+ viable cell density is greater than 0.5×106 cells/mL, and wherein no culture medium is added at the second time point if CD45+ viable cell density is less than 0.5×106 cells/mL,
• wherein a whole culture volume (1×) of cell culture medium is added at the third time point if CD45+ viable cell density is greater than 0.5×106 cells/mL, and wherein a half culture volume (0.5×) of cell culture medium is added at the third time point if CD45+ viable cell density is less than 0.5×106 cells/mL,
• wherein step (b) is from about 10 to about 13 days or from about 11 to about 13 days, optionally wherein the first time point is between about day 5 and about day 7, the second time point is between about day 7 and about day 9, and the third time point is between about day 10 and about day 12, and
• optionally wherein the method proceeds to step (c) instead of adding the cell culture medium at the third time point if there are greater than about 20×106 CD3+ cells/mL, optionally wherein the third time point is day 11.

The method of any preceding claim, wherein the first population of TILs that is cultured in step (c) comprises between about 0.75×106 cells and about 20×106 cells or between about 1×106 cells and about 20×106 cells,

• wherein the second cell culture medium comprises about 2000 IU/mL of IL-2,
• wherein the second cell culture medium comprises human AB serum but does not comprise FBS,
• wherein the expansion in step (c) comprises a static expansion followed by a dynamic expansion,
• wherein the static expansion is performed in a 3 L bag, optionally wherein the bag is a ethylene vinyl acetate (EVA) bag or a fluorinated ethylene propylene (FEP) bag,
• wherein the static expansion is for about 5 to about 7 days, and wherein the dynamic expansion is for about 7 to about 9 days,
• wherein the static expansion is performed in a working volume of about 1500 mL to about 2500 mL or about 1750 mL to about 2250 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 500 mL to about 750 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the static expansion is performed in a working volume of about 2000 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 625 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the dynamic expansion is performed in a working volume of about 2400 mL to about 4000 mL or about 2800 mL to about 3600 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 500 mL to about 1500 mL or about 750 mL to about 1250 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the dynamic expansion is performed in a working volume of about 3200 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 1000 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the dynamic expansion comprises rocking the first population of cells at a rocking angle of about 8 degrees,
• wherein the dynamic expansion comprises perfusion steps at fourth, fifth, and sixth time points, wherein the perfusion comprises removing spent cell culture medium while simultaneously adding fresh cell culture medium in equal parts to maintain a constant culture volume,
• wherein the fourth time point is days 20-21, wherein the fifth time point is days 22-23, and wherein the sixth time point is days 24-30,
• wherein the perfusion at the fourth time point is about 0.6 to about 1 L/day, the perfusion at the fifth time point is about 1.4 to about 1.8 L/day, and the perfusion at the sixth time point is about 3 to about 3.4 L/day if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the perfusion at the fourth time point is about 0.2 to about 0.3 L/day, the perfusion at the fifth time point is about 0.4 to about 0.6 L/day, and the perfusion at the sixth time point is about 0.8 to about 1.2 L/day if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the perfusion at the fourth time point is about 0.8 L/day, the perfusion at the fifth time point is about 1.6 L/day, and the perfusion at the sixth time point is about 3.2 L/day if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the perfusion at the fourth time point is about 0.25 L/day, the perfusion at the fifth time point is about 0.5 L/day, and the perfusion at the sixth time point is about 1 L/day if the first population of TILs is between about 0.75×106 cells and about 3×106 cells.

The method of any preceding claim, wherein the first cell culture medium comprises fetal bovine serum (FBS) and about 2000 IU/mL of IL-2,

• wherein step (b) comprises initially seeding the processed resected tumor product in about 20 to about 40 mL of the cell culture medium in a 70-mL cell culture bag,
• wherein step (b) comprises initially seeding the processed resected tumor product in a whole culture volume of cell culture medium, adding the cell culture medium at a first time point, conditionally adding the cell culture medium at a second time point based on cell concentration, and optionally conditionally adding the cell culture medium at a third time point based on cell concentration,
• wherein a half culture volume (0.5×) of cell culture medium is added at the first time point,
• wherein adding the cell culture medium at the second and third time points is determined based on CD45+ viable cell density,
• wherein a half culture volume (0.5×) of cell culture medium is added at the second time point if CD45+ viable cell density is greater than 0.5×106 cells/mL, and wherein no culture medium is added at the second time point if CD45+ viable cell density is less than 0.5×106 cells/mL,
• wherein a whole culture volume (1×) of cell culture medium is added at the third time point if CD45+ viable cell density is greater than 0.5×106 cells/mL, and wherein a half culture volume (0.5×) of cell culture medium is added at the third time point if CD45+ viable cell density is less than 0.5×106 cells/mL,
• wherein step (b) is from about 10 to about 13 days or from about 11 to about 13 days, optionally wherein the first time point is between about day 5 and about day 7, the second time point is between about day 7 and about day 9, and the third time point is between about day 10 and about day 12, and
• optionally wherein the method proceeds to step (c) instead of adding the cell culture medium at the third time point if there are greater than about 20×106 CD3+ cells/mL, optionally wherein the third time point is day 11,
• wherein the first population of TILs that is cultured in step (c) comprises between about 0.75×106 cells and about 20×106 cells or between about 1×106 cells and about 20×106 cells,
• wherein the second cell culture medium comprises about 2000 IU/mL of IL-2,
• wherein the second cell culture medium comprises human AB serum but does not comprise FBS,
• wherein the expansion in step (c) comprises a static expansion followed by a dynamic expansion,
• wherein the static expansion is performed in a 3 L bag, optionally wherein the bag is a ethylene vinyl acetate (EVA) bag or a fluorinated ethylene propylene (FEP) bag,
• wherein the static expansion is for about 5 to about 7 days, and wherein the dynamic expansion is for about 7 to about 9 days,
• wherein the static expansion is performed in a working volume of about 1500 mL to about 2500 mL or about 1750 mL to about 2250 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 500 mL to about 750 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the static expansion is performed in a working volume of about 2000 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 625 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the dynamic expansion is performed in a working volume of about 2400 mL to about 4000 mL or about 2800 mL to about 3600 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 500 mL to about 1500 mL or about 750 mL to about 1250 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the dynamic expansion is performed in a working volume of about 3200 mL if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the static expansion is performed in a working volume of about 1000 mL if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the dynamic expansion comprises rocking the first population of cells at a rocking angle of about 8 degrees,
• wherein the dynamic expansion comprises perfusion steps at fourth, fifth, and sixth time points, wherein the perfusion comprises removing spent cell culture medium while simultaneously adding fresh cell culture medium in equal parts to maintain a constant culture volume,
• wherein the fourth time point is days 20-21, wherein the fifth time point is days 22-23, and wherein the sixth time point is days 24-30,
• wherein the perfusion at the fourth time point is about 0.6 to about 1 L/day, the perfusion at the fifth time point is about 1.4 to about 1.8 L/day, and the perfusion at the sixth time point is about 3 to about 3.4 L/day if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the perfusion at the fourth time point is about 0.2 to about 0.3 L/day, the perfusion at the fifth time point is about 0.4 to about 0.6 L/day, and the perfusion at the sixth time point is about 0.8 to about 1.2 L/day if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the perfusion at the fourth time point is about 0.8 L/day, the perfusion at the fifth time point is about 1.6 L/day, and the perfusion at the sixth time point is about 3.2 L/day if the first population of TILs is between about 3×106 cells and about 20×106 cells, and wherein the perfusion at the fourth time point is about 0.25 L/day, the perfusion at the fifth time point is about 0.5 L/day, and the perfusion at the sixth time point is about 1 L/day if the first population of TILs is between about 0.75×106 cells and about 3×106 cells,
• wherein the harvesting is performed using a harvest media comprising about 5% human serum albumin (HSA) and PBS,
• wherein the TIL formulation in step (d) comprises about 2.5% HSA and about 5% DMSO, and
• wherein the formulating comprises adding HSA and DMSO to the second population of TILs in a closed system.