Processes for generating til products using pd-1/tigit talen double knockdown

A method for preparing expanded tumor infiltrating lymphocytes (TILs) having reduced expression of PD-1 and TIGIT, comprising:

• (a) obtaining a first population of TILs from a tumor sample resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments, or from a tumor sample obtained from a patient by surgical resection, needle biopsy, core biopsy, small biopsy, or other means;
• (b) culturing the first population of TILs in a first cell culture medium comprising IL-2 and IL-21 for about 5-7 days to produce a second population of TILs, optionally wherein the first cell culture medium comprising IL-2 and IL-21 is replaced on the 3rd day, the 4th day, or the 5th day of step (b);
• (c) activating the second population of TILs for about 2-4 days, to produce a third population of TILs;
• (d) introducing a first TALE nuclease (TALEN) system targeting a first gene selected from the group consisting of a gene encoding PD-1 and a gene encoding TIGIT into at least a portion of the third population of TILs, to produce a fourth population of TILs;
• (e) resting the fourth population of TILs in the first cell culture medium comprising IL-2 for about 2 to 3 days;
• (f) introducing a second TALEN system targeting a second gene selected from the group consisting of the gene encoding PD-1 and the gene encoding TIGIT into at least a portion of the fourth population of TILs to produce a fifth population of TILs, wherein the first gene and the second gene are different;
• and
• (g) culturing the fifth population of TILs in a second cell culture medium comprising IL-15 and IL-21, antigen presenting cells (APCs), and OKT-3 for about 7-11 days, to produce sixth population of TILs having reduced expression of the first gene and the second gene, optionally wherein the second cell culture medium comprising IL-15 and IL-21 is replaced on the 3rd day, the 4th day, the 5th day, the 6th day, the 7th day, or the 8th day of step (g).

A method for treating a cancer patient, comprising:

• (a) obtaining a first population of TILs from a tumor sample resected from the cancer patient by processing a tumor sample obtained from the cancer patient into multiple tumor fragments, or from a tumor sample obtained from the cancer patient by surgical resection, needle biopsy, core biopsy, small biopsy, or other means;
• (b) culturing the first population of TILs in a first cell culture medium comprising IL-2 and IL-21 for about 5-7 days to produce a second population of TILs, optionally wherein the first cell culture medium comprising IL-2 and IL-21 is replaced on the 3rd day, the 4th day, or the 5th day of step (b);
• (c) activating the second population of TILs for about 2-4 days, to produce a third population of TILs;
• (d) introducing a first TALE nuclease (TALEN) system targeting a first gene selected from the group consisting of a gene encoding PD-1 and a gene encoding TIGIT into at least a portion of the third population of TILs, to produce a fourth population of TILs;
• (e) resting the fourth population of TILs in the first cell culture medium comprising IL-2 and IL-21 for about 2 to 3 days;
• (f) introducing a second TALEN system targeting a second gene selected from the group consisting of the gene encoding PD-1 and the gene encoding TIGIT into at least a portion of the fourth population of TILs to produce a fifth population of TILs, wherein the first gene and the second gene are different;
• (g) culturing the fifth population of TILs in a second cell culture medium comprising IL-15 and IL-21, antigen presenting cells (APCs), and OKT-3 for about 7-11 days, to produce sixth population of TILs having reduced expression of the first gene and the second gene, optionally wherein the second cell culture medium comprising IL-15 and IL-21 is replaced on the 3rd day, the 4th day, the 5th day, the 6th day, the 7th day, or the 8th day of step (g);
• and
• (h) administering a therapeutically effective dosage of the sixth population of TILs to the cancer patient.