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 comprising:
(a) obtaining a first population of TILs from a tumor resected from a patient by processing a tumor sample obtained from the patient into multiple tumor fragments;
(b) adding the tumor fragments into a closed system;
(c) performing a first expansion by culturing the first population of TILs in a cell culture medium comprising IL-2, and optionally OKT-3, 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 about 3-14 days to obtain the second population of TILs, wherein the transition from step (b) to step (c) occurs without opening the system;
(d) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, optionally OKT-3, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the second expansion is performed for about 4-6 days to obtain the third 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 (c) to step (d) occurs without opening the system;
(e) dividing the third population of TILs into a first plurality of 2-5 subpopulations of TILs, wherein at least 1.0×10 9 TILs are present in each subpopulation, wherein the transition from step (d) to (e) occurs without opening the system;
(f) performing a third expansion of the first plurality of subpopulations of TILs by supplementing the cell culture medium of each subpopulation of TILs with additional IL-2, optionally OKT-3, to produce a second plurality of subpopulations of TILs, wherein the third expansion is performed for about 5-7 days, wherein the third expansion for each subpopulation is performed in a closed container providing a third gas-permeable surface area, and wherein the transition from step (e) to step (f) occurs without opening the system;
(g) harvesting the second plurality of subpopulations of TILs obtained from step (f), wherein the transition from step (f) to step (g) occurs without opening the system; and
(h) transferring the harvested subpopulations of TILs from step (g) to one or more infusion bags, wherein the transition from step (g) to (h) occurs without opening the system.
2. The method according to claim 1 , further comprising the step of cryopreserving the infusion bag comprising the harvested TIL population in step (f) using a cryopreservation process.
3. The method according to claim 1 , wherein the cryopreservation process is performed using a 1:1 ratio of harvested TIL population to cryopreservation media.
4. The method according to claim 1 , wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).
5. The method according to claim 4 , wherein the PBMCs are irradiated and allogeneic.
6. The method according to claim 1 , wherein the antigen-presenting cells are artificial antigen-presenting cells.
7. The method according to claim 1 , wherein the cell culture medium in step (d) and/or step (f) further comprises IL-15 and/or IL-21.
8. The method according to claim 1 , wherein the IL-2 concentration is about 10,000 IU/mL to about 5,000 IU/mL.
9. The method according to claim 7 , wherein the IL-15 concentration is about 500 IU/mL to about 100 IU/mL.
10. The method according to claim 1 , wherein the IL-21 concentration is about 20 IU/mL to about 0.5 IU/mL.
11. The method according to claim 1 , wherein the infusion bags in step (h) are HypoThermosol-containing infusion bags.
12. The method according to claim 3 , wherein the cryopreservation media comprises dimethlysulfoxide (DMSO).
13. The method according to claim 9 , wherein the wherein the cryopreservation media comprises 7% to 10% DMSO.
14. The method according to claim 1 , wherein the first period in step (c) is performed within a period of 10 days, 11 days, or 12 days.
15. The method according to claim 1 , wherein the first period in step (c) is performed within a period of 11 days.
16. The method according to claim 1 , wherein steps (a) through (h) are performed within a period of about 10 days to about 22 days.
17. The method according to claim 1 , wherein steps (a) through (h) are performed within a period of about 20 days to about 22 days.
18. The method according to claim 1 , wherein steps (a) through (h) are performed within a period of about 15 days to about 20 days.
19. The method according to claim 1 , wherein steps (a) through (h) are performed within a period of about 10 days to about 20 days.
20. The method according to claim 1 , wherein the second plurality of subpopulations of TILs harvested in step (g) comprises sufficient TILs for a therapeutically effective dosage of the TILs.
21. The method according to claim 20 , wherein the number of TILs sufficient for a therapeutically effective dosage is from about 2.3×10 10 to about 13.7×10 10 .
22. The method according to claim 1 , wherein the third population of TILs in step (d) provides for increased efficacy, increased interferon-gamma production, increased polyclonality, increased average IP-10, and/or increased average MCP-1 when administered to a subject.
23. The method according to claim 1 , wherein the second plurality of subpopulations of TILs in step (df) provides for at least a five-fold or more interferon-gamma production when administered to a subject.
24. The method according to claim 1 , wherein the second plurality of subpopulations of TILs in step (f) is a therapeutic population of TILs which comprises an increased subpopulation of effector T cells and/or central memory T cells relative to the second population of TILs, wherein the effector T cells and/or central memory T cells in the therapeutic population of TILs exhibit one or more characteristics selected from the group consisting of expressing CD27+, expressing CD28+, longer telomeres, increased CD57 expression, and decreased CD56 expression relative to effector T cells, and/or central memory T cells obtained from the second population of cells.
25. The method according to claim 1 , wherein the effector T cells and/or central memory T cells obtained from the second plurality of subpopulations of TILs in step (f) exhibit increased CD57 expression and decreased CD56 expression relative to effector T cells and/or central memory T cells obtained from the second population of cells.
26. The method according to claim 1 , wherein the risk of microbial contamination is reduced as compared to an open system.
27. The method according to claim 1 , wherein the TILs from step (h) are infused into a patient.
28. The method for expanding TILs according to claim 1 , further comprising between steps (c) and (d) a step of determining the number of TILs in said second population of TILs in step (c) and removing excess TILs from said second population, wherein said second population has had more than 2×10 8 TILs before the removing, and said second population of TILs has at most 2×10 8 TILs after the removing, and wherein the step occurs without opening the system.
29. The method for expanding TILs according to claim 1 , wherein supplementing the cell culture medium of the second population of TILs in step (d) further comprises supplementing said cell culture medium with a tumor necrosis factor receptor superfamily (TNFRSF) agonist.
30. The method for expanding TILs according to claim 1 , wherein supplementing the cell culture medium of each subpopulation of TILs in step (f) further comprises supplementing said cell culture medium with a tumor necrosis factor receptor superfamily (TNFRSF) agonist.