Processes for production of tumor infiltrating lymphocytes and uses of same in immunotherapy
The present invention provides improved and/or shortened methods for expanding TILs and producing therapeutic populations of TILs, including novel methods for expanding TIL populations in a closed system that lead to improved efficacy, improved phenotype, and increased metabolic health of the TILs in a shorter time period, while allowing for reduced microbial contamination as well as decreased costs. Such TILs find use in therapeutic treatment regimens.
1. A method for expanding tumor infiltrating lymphocytes (TILs) into a therapeutic population of TILs, the method comprising:
(a) adding a tumor digest or tumor fragments into a closed system, wherein the tumor digest or tumor fragments comprise a first population of TILs and are obtained from a tumor that was resected from a subject;
(b) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2 to produce a second population of TILs, wherein the first expansion is performed in a closed container providing a first gas-permeable surface area, wherein the first expansion is performed for a first period of about 3 to 11 days to obtain the second population of TILs, and wherein the transition from step (a) to step (b) occurs without opening the system;
(c) performing a second expansion by supplementing the cell culture medium with additional IL-2, OKT-3, and antigen presenting cells (APCs) to produce a third population of TILs, wherein the second expansion is performed for a second period of about 7 to 11 days to obtain the third population of TILs, wherein the third population of TILs is a therapeutic population of TILs, wherein the second expansion is performed in a closed container providing a second gas-permeable surface area, and wherein the transition from step (b) to step (c) occurs without opening the system;
(d) harvesting the therapeutic population of TILs obtained from step (c), wherein the transition from step (c) to step (d) occurs without opening the system;
(e) transferring the harvested therapeutic population of TILs from step (d) to an infusion bag, wherein the transfer from step (d) to (e) occurs without opening the system; and
(f) cryopreserving the infusion bag comprising the harvested therapeutic population of TILs from step (e) using a cryopreservation process.
2. The method according to claim 1 , wherein the tumor digest in step (a) was prepared by incubating a sample of the tumor that was resected from the subject in an enzymatic media.
3. The method according to claim 2 , further comprising disrupting the tumor sample mechanically so as to dissociate the tumor sample.
4. The method according to claim 3 , further comprising purifying the disassociated tumor sample using a density gradient separation.
5. The method according to claim 2 , wherein the enzymatic media comprises DNase.
6. The method according to claim 5 , wherein the enzymatic media comprises 30 units/mL of DNase.
7. The method according to claim 2 , wherein the enzymatic media comprises collagenase.
8. The method according to claim 7 , wherein the enzymatic media comprises 1.0 mg/mL of collagenase.
9. The method according to claim 1 , wherein the medium in the first expansion and/or the second expansion is free of human serum.
10. The method according to claim 1 , wherein the therapeutic population of TILs harvested in step (d) comprises sufficient TILs for use in administering a therapeutically effective dosage to a subject.
11. The method according to claim 10 , wherein the number of TILs sufficient for administering a therapeutically effective dosage is from about 1×10 9 to about 9×10 10 .
12. The method according to claim 1 , wherein the APCs are peripheral blood mononuclear cells (PBMCs).
13. The method according to claim 12 , wherein the PBMCs are supplemented at a ratio of about 1:25 TIL:PBMCs.
14. The method according to claim 1 , wherein the therapeutic population of TILs harvested in step (d) exhibits an increased subpopulation of CD8+ cells relative to the first and/or second population of TILs.
15. The method according to claim 1 , wherein the first expansion in step (b) and the second expansion in step (c) are each individually performed within a period of 11 days.
16. The method according to claim 1 , wherein steps (a) through (e) are performed in about 10 days to about 22 days.
17. The method according to claim 1 , wherein steps (a) through (e) are performed in about 15 days to about 22 days.
18. The method according to claim 1 , wherein steps (a) through (e) are performed in about 20 days to about 22 days.
19. The method according to claim 1 , wherein the tumor digest is a cryopreserved tumor digest.