Method for producing tumor-infiltrating lymphocytes and their use in immunotherapy

Abstract

Providing a method for producing tumor-infiltrating lymphocytes and their use in immunotherapy. [Solution] The present invention provides an improved and / or shortened method for expanding TIL populations and generating therapeutic TIL populations, including a novel method for expanding TIL populations in a closed system, which enables improved efficacy, improved phenotype, and increased metabolic health of TILs in a shorter period of time, while reducing microbial contamination and costs. Such TILs find use in therapeutic treatment regimens.

Claims

1. A method for expanding tumor-infiltrating lymphocytes (TILs) into a therapeutic TIL population, comprising: (a) adding processed tumor fragments from a tumor resected from a patient to a closed system to obtain a first TIL population; (b) performing a first expansion culture to produce a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optionally OKT-3, wherein the first expansion culture is performed in a closed container providing a first gas-permeable surface area, the first expansion culture is performed for about 3 to 14 days to obtain the second TIL population, the second TIL population being at least 50 times larger in number than the first TIL population, and the transition from step (a) to step (b) occurs without opening the system; (c) adding additional IL-2, optionally OKT-3, to the cell culture medium of the second TIL population. A method comprising: (d) recovering the therapeutic TIL population obtained from step (c), the transition from step (c) to step (d), the transition from step (d) to step (e), the transfer of the recovered TIL population from step (d) to an infusion bag, the transition from step (d) to step (e), the transfer of the transferred TIL population from step (d) to an infusion bag, the transition from step (d) to step (e), the transfer of the transferred TIL population from step (d) to an infusion bag.

2. The method according to claim 1, wherein the therapeutic TIL population recovered in step (d) contains enough TILs to make a therapeutically effective dose of TILs.

3. The number of TILs sufficient for a therapeutically effective dosage is approximately 2.3 × 10⁻⁶ １０ ~Approx. 13.7×10 １０ The method according to claim 2, wherein there is one.

4. The method according to claim 3, further comprising the step of freezing and preserving the infusion bag containing the recovered TIL population using a cryopreservation process.

5. The method according to claim 4, wherein the cryopreservation process is carried out using a 1:1 ratio of the recovered TIL population to the cryopreservation medium.

6. The method according to claim 5, wherein the antigen-presenting cells are peripheral blood mononuclear cells (PBMCs).

7. The method according to claim 6, wherein the PBMC is irradiated and is of the same type but different in nature.

8. The method according to claim 7, wherein the PBMC is added to the cell culture on any of days 9 to 14 in step (c).

9. The method according to claim 1, wherein the antigen-presenting cells are artificial antigen-presenting cells.

10. The method according to claim 1, wherein the recovery in step (d) is carried out using a LOVO cell processing system.

11. The aforementioned multiple fragments consist of approximately 4 to 50 fragments, and each fragment is approximately 27 mm in length. ３ The method according to claim 1, having the volume of

12. The aforementioned multiple fragments consist of approximately 30 to 60 fragments, and the total volume is approximately 1300 mm³. ３ ~Approx. 1500mm ３ The method according to claim 1.

13. The aforementioned multiple fragments consist of approximately 50 fragments, and the total volume is approximately 1350 mm³. ３ The method according to claim 2.

14. The method according to claim 1, wherein the plurality of fragments comprises about 50 fragments, and the total mass is about 1 gram to about 1.5 grams.

15. The method according to claim 1, wherein the plurality of fragments include about four fragments.

16. The method according to claim 1, wherein the second cell culture medium is provided in a container selected from the group consisting of G containers and Xuri cell culture bags.

17. The method according to claim 1, wherein the infusion bag in step (e) is a HypoThermosol-containing infusion bag.

18. The method according to claim 1, wherein the first period of step (b) and the second period of step (c) are carried out individually within periods of 10, 11, or 12 days, respectively.

19. The method according to claim 1, wherein the first period of step (b) and the second period of step (c) are each carried out individually within a period of 11 days.

20. Steps (a) to (e) are carried out within a period of approximately 10 to 22 days, according to the method of claim 1.

21. The method according to claim 1, wherein steps (a) to (e) are carried out within a period of approximately 10 to 20 days.

22. Steps (a) to (e) are carried out within a period of approximately 10 to 15 days, according to the method of claim 1.

23. Steps (a) to (e) are carried out within 22 days, according to the method of claim 1.

24. The method according to claim 4, wherein steps (a) to (e) and cryopreservation are carried out within 22 days.

25. The method according to any one of claims 1 to 24, wherein steps (b) to (e) are performed in a single container, and performing steps (b) to (e) in a single container results in an increased TIL yield per excised tumor compared to performing steps (b) to (e) in two or more containers.

26. The method according to any one of claims 1 to 25, wherein the antigen-presenting cells are added to the TIL during the second period of step (c) without opening the system.

27. The method according to any one of claims 1 to 26, wherein the third TIL population of step (d) is a therapeutic TIL population comprising an increased subpopulation of effector T cells and / or central memory T cells compared to the second TIL population, and the effector T cells and / or central memory T cells obtained in the therapeutic TIL population exhibit one or more characteristics selected from the group consisting of CD27+ expression, CD28+ expression, longer telomeres, increased CD57 expression, and decreased CD56 expression, compared to the effector T cells and / or central memory T cells obtained from the second cell population.

28. The method according to any one of claims 1 to 27, wherein the effector T cells and / or central memory T cells obtained in the therapeutic TIL population exhibit increased CD57 expression and decreased CD56 expression compared to the effector T cells and / or central memory T cells obtained from the second cell population.

29. The method according to any one of claims 1 to 28, wherein the risk of microbial contamination is reduced compared to an open system.

30. The method according to any one of claims 1 to 29, wherein the TIL from step (e) is injected into the patient.

31. The method according to any one of claims 1 to 30, wherein the closed container comprises a single bioreactor.

32. The method according to claim 31, wherein the closed container includes G-REX-10.

33. The method according to claim 31, wherein the closed container includes G-REX-100.

34. The method according to any one of claims 1 to 33, wherein in step (d), the antigen-presenting cells (APCs) are added to the cell culture of the second TIL population in an APC:TIL ratio of 25:1 to 100:

1.

35. The cell culture is 2.5 × 10 ９ Individual APCs vs 100 x 10 ６ The method according to claim 34, having a ratio of TILs.

36. The method according to any one of claims 1 to 33, wherein in step (c), the antigen-presenting cells (APCs) are added to the cell culture of the second TIL population in an APC:TIL ratio of 25:1 to 100:

1.

37. The cell culture has a ratio of 2.5×10 ９ APC per 100×10 ６ TIL, the method according to claim 36.

38. A wide-culture TIL population for use in the treatment of subjects with cancer, comprising: (a) obtaining a first TIL population from a tumor excised from a subject by processing a tumor sample obtained from a patient into multiple tumor fragments; (b) adding the tumor fragments to a closed system; (c) performing a first wide-culture to produce a second TIL population by culturing the first TIL population in a cell culture medium containing IL-2 and optionally OKT-3, wherein the first wide-culture is performed in a closed container providing a first gas-permeable surface area, the first wide-culture is performed for about 3 to 14 days to obtain the second TIL population, the second TIL population is at least 50 times larger in number than the first TIL population, and the transition from step (b) to step (c) occurs without opening the system; (d) performing a second wide-culture by adding additional IL-2, optionally OKT-3 and antigen-presenting cells (APCs) to the cell culture medium of the second TIL population. A third TIL population, which is a grown-up cultured TIL population, can be obtained by a method comprising: (c) generating a third TIL population, wherein the second expansion culture is carried out over a period of about 7 to 14 days to obtain the third TIL population, the third TIL population is a therapeutic TIL population, the second expansion culture is carried out in a closed container providing a second gas-permeable surface area, the transition from step (c) to step (d) occurs without opening the system; (e) recovering the therapeutic TIL population obtained from step (d), the transition from step (d) to step (e) occurs without opening the system; and (f) transferring the recovered TIL population from step (e) to an infusion bag, the transition from step (e) to (f) occurs without opening the system; and (g) optionally, freezing the infusion bag containing the recovered TIL population from step (f) using a cryopreservation process.

39. The method for generating the TIL further comprises one or more features of any one of claims 1 to 37, the TIL group according to claim 38.

40. A cryopreservation composition comprising the TIL group described in claim 38, a cryoprotective medium containing dimethyl sulfoxide (DMSO), and an electrolyte solution.

41. The cryopreservation composition according to claim 40, further comprising one or more stabilizers and one or more lymphocyte growth factors.

42. The cryopreservation composition according to claim 41, wherein the one or more stabilizers comprises human serum albumin (HSA), and the one or more lymphocyte growth factors comprises IL-2.

43. The cryopreservation composition according to claim 42, optionally comprising OKT-3.

44. The cryopreservation composition according to claim 40, wherein the cryoprotective medium containing DMSO and the electrolyte solution are present in a ratio of about 1.1:1 to about 1:1.

1.

45. The cryopreservation composition according to claim 44, wherein the cryoprotective medium containing DMSO and the electrolyte solution are present in a ratio of about 1:

1.

46. 100 mL of the above composition contains approximately 1 × 10 ６ ~Approx. 9×10 １４ The cryopreservation composition according to claim 40, comprising: a TIL population in an amount of 1; a cryoprotective medium containing DMSO in an amount of about 30 mL to about 70 mL; an electrolyte solution in an amount of about 30 mL to about 70 mL; an HSA in an amount of about 0.1 g to about 1.0 g; and an IL-2 in an amount of about 0.001 mg to about 0.005 mg.

47. 100 mL of the above composition contains approximately 1 × 10 ７ ~Approx. 1×10 １１ The cryopreservation composition according to claim 40, comprising: an amount of the TIL population; an amount of about 30 mL to about 70 mL of the cryoprotective medium containing about 10% DMSO; an amount of about 30 mL to about 70 mL of the electrolyte solution; an amount of about 0.3 g to about 0.7 g of HSA; and an amount of about 0.001 mg to about 0.003 mg of IL-2.

48. 100 mL of the above composition contains approximately 1 × 10 ７ ~Approx. 1×10 １１ The aforementioned TIL population in quantities of 1; approximately 10% DMSO The cryopreservation composition according to claim 40, comprising essentially about 30 mL to about 70 mL of the cryoprotective medium; about 30 mL to about 70 mL of the electrolyte solution; about 0.3 g to about 0.7 g of HSA; and about 0.001 mg to about 0.003 mg of IL-2.

49. The cryopreservation composition according to claim 40, for use in treating subjects having cancer.

50. A cryopreservation composition comprising a TIL group, a cryoprotective medium containing dimethyl sulfoxide (DMSO), and an electrolyte solution, wherein 100 mL of the composition contains about 1 × 10⁶ ７ ~Approx. 1×10 １１ A cryopreservation composition comprising: a certain amount of the TIL population; about 30 mL to about 70 mL of the cryoprotective medium containing about 10% DMSO; about 30 mL to about 70 mL of the electrolyte solution; about 0.3 g to about 0.7 g of HSA; and about 0.001 mg to about 0.003 mg of IL-2.

51. A cryopreservation composition comprising a TIL group, a cryoprotective medium containing dimethyl sulfoxide (DMSO), and an electrolyte solution, wherein 100 mL of the composition contains about 1 × 10⁶ units. ７ ~Approx. 1×10 １１ A cryopreservation composition comprising: an amount of the TIL population; an amount of the cryoprotective medium in the form of about 30 mL to about 70 mL, essentially consisting of about 10% DMSO; an amount of the electrolyte solution in the form of about 30 mL to about 70 mL; an amount of HSA in the form of about 0.3 g to about 0.7 g; and an amount of IL-2 in the form of about 0.001 mg to about 0.003 mg.

52. An infusion bag comprising the cryopreservation composition according to any one of claims 40 to 51.

53. The infusion bag according to claim 52, which is a hypothermol-containing infusion bag.

54. A storage bag comprising the cryopreservation composition according to any one of claims 40 to 51.

55. The storage bag according to claim 54, which is a CryoStore CS750 freezer bag.

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