Method for producing TIL products enriched with tumor antigen specific T cells

文档序号：1026666 发布日期：2020-10-27 浏览：3次中文

阅读说明：本技术 产生富含肿瘤抗原特异性t细胞的til产品的方法 (Method for producing TIL products enriched with tumor antigen specific T cells ) 是由 C·沙尔捷-库尔托 K·里特皮柴于 2019-01-08 设计创作，主要内容包括：本发明提供了重编程TIL的改进和/或缩短的过程和方法,以制备具有增加的治疗功效的治疗性TIL群。此种重编程的TIL可用于治疗方案中。(The present invention provides improved and/or shortened processes and methods for reprogramming TILs to produce therapeutic TIL populations with increased therapeutic efficacy. Such reprogrammed TILs may be used in treatment protocols.)

1. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(i) obtaining a first TIL population from a resected tumor of a patient;

(ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population;

(iii) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cell APCs, to produce a third TIL population; the number of third TIL groups is at least 100 times greater than the number of second TIL groups; performing a second amplification for at least 14 days to obtain a third TIL population; the third TIL population is a therapeutic TIL population; and

(iv) Exposing the second and/or third TIL populations to the transcription factor TF and/or other molecule capable of transiently altering protein expression; the TF and/or other molecule capable of transiently altering protein expression provides for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population.

2. The method of claim 1, wherein the method further comprises:

(v) (iii) performing a further second expansion by supplementing the cell culture medium of the third TIL population with further IL-2, further OKT-3 and further APCs, before or after step (iv); (iv) said further second expansion is carried out for at least 14 days to obtain a larger population of therapeutic TILs than the population of therapeutic TILs obtained in step (iii); the larger therapeutic TIL population exhibits altered numbers of T cells specific for tumor antigens.

3. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population; the first amplification is carried out in a closed vessel providing a first gas permeable surface region; performing the first amplification for about 3 to 14 days to obtain a second TIL population; the number of second TIL groups is at least 50 times greater than the number of first TIL groups; 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 TIL population with additional IL-2, OKT-3 and antigen presenting cell APCs, to produce a third TIL population; performing a second amplification for about 7 to 14 days to obtain a third TIL population; the third TIL population is a therapeutic TIL population; the second amplification is carried out in a closed vessel providing a second gas permeable surface region; the transition from step (c) to step (d) occurs without opening the system;

(e) exposing the second and/or third TIL populations to the transcription factor TF and/or other molecule capable of transiently altering protein expression; the TF and/or other molecule capable of transiently altering protein expression provides for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population;

(f) harvesting the therapeutic TIL population obtained from step (d); the transition from step (d) to step (e) occurs without opening the system; and

(g) transferring the TIL population harvested from step (e) to an infusion bag; the transition from step (e) to step (f) occurs without opening the system.

4. The method of claim 3, wherein the method further comprises the steps of: cryopreserving the infusion bag comprising the TIL population harvested from step (f) using a cryopreservation method.

5. The method of claim 4, wherein the cryopreservation method uses a 1: the harvested TIL population at a ratio of 1 was performed on cryopreservation media.

6. The method according to claim 4, wherein the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

7. The method of claim 6, wherein the PBMCs are irradiated and allogeneic.

8. The method of claim 6, wherein the PBMCs are added to the cell culture on any of days 9 to 14 of step (d).

9. The method of claim 6, wherein the antigen presenting cell is an artificial antigen presenting cell.

10. The method of claim 3, wherein the harvesting in step (e) is performed using a membrane-based cell processing system.

11. The method of claim 3, wherein the harvesting in step (e) is performed using a LOVO cell processing system.

12. The method of claim 3, wherein the plurality of fragments comprises about 4 to about 50 fragments, each fragment having a volume of about 27mm³。

13. The method of claim 3, wherein the plurality of fragments comprises about 30 to about 60 fragments having a total volume of about 1300mm ³To about 1500mm³。

14. The method of claim 13, wherein the plurality of fragments comprises about 50 fragments having a total volume of about 1350mm³。

15. The method of claim 1, wherein the plurality of chips comprises about 50 chips having a total mass of about 1 gram to about 1.5 grams.

16. The method of claim 3, wherein the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

17. The method of claim 3, wherein the cell culture medium in step (d) further comprises IL-15 and/or IL-21.

18. The method of any one of claims 1-17, wherein the IL-2 concentration is about 10,000IU/mL to about 5,000 IU/mL.

19. The method of claim 17, wherein the IL-15 concentration is about 500IU/mL to about 100 IU/mL.

20. The method of claim 17, wherein the IL-21 concentration is about 20IU/mL to about 0.5 IU/mL.

21. A method according to claim 3, wherein the infusion bag in step (f) is a hypo thermosol-containing infusion bag.

22. The method of claim 5, wherein the cryopreservation media comprises dimethyl sulfoxide (DMSO).

23. The method of claim 22, wherein the cryopreservation media comprises 7% to 10% DMSO.

24. The method of claim 3, wherein the first stage in step (c) and the second stage in step (e) are performed for a period of 10 days, 11 days, or 12 days, respectively.

25. The method of claim 3, wherein the first stage in step (c) and the second stage in step (e) are each performed for a period of 11 days.

26. The method of claim 3, wherein steps (a) through (f) are performed for a period of about 10 days to about 22 days.

27. The method of claim 3, wherein steps (a) through (f) are performed for a period of about 20 days to about 22 days.

28. The method of claim 3, wherein steps (a) through (f) are performed for a period of about 15 days to about 20 days.

29. The method of claim 3, wherein steps (a) through (f) are performed for a period of about 10 days to about 20 days.

30. The method of claim 3, wherein steps (a) through (f) are performed for a period of about 10 days to about 15 days.

31. The method of claim 3, wherein steps (a) through (f) are performed for less than 22 days.

32. The method of claim 3, wherein steps (a) through (f) are performed for 20 days or less.

33. The method of claim 3, wherein steps (a) through (f) are performed for less than 15 days.

34. The method of claim 3, wherein steps (a) through (f) are performed for 10 days or less.

35. The method of claim 5, wherein steps (a) through (f) and cryopreservation are performed for less than 22 days.

36. The method of any one of claims 3 to 35, wherein the therapeutic TIL population harvested in step (e) comprises TIL sufficient for a therapeutically effective dose of TIL.

37. The method of claim 36, wherein the amount of TIL sufficient for a therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

38. The method of any one of claims 3 to 37, wherein steps (b) to (e) are performed in a single vessel; performing steps (b) to (e) in a single vessel results in an increased TIL production per resected tumor as compared to performing steps (b) to (e) in more than one vessel.

39. A method according to any one of claims 3 to 38, wherein, during the second stage of step (d), antigen presenting cells are added to the TIL without turning the system on.

40. The method of any one of claims 3 to 39, wherein the third TIL population in step (d) provides at least 5-fold or greater interferon- γ production when administered to a subject.

41. The method of any one of claims 3 to 40, wherein the risk of microbial contamination is reduced compared to an open system.

42. The method of any one of claims 3 to 41, wherein the TIL from step (f) or step (g) is injected into the patient.

43. The method of any one of claims 3 to 42, wherein the plurality of fragments comprises about 4 fragments.

44. A method of treating a subject having cancer, the method comprising administering expanded tumor infiltrating lymphocyte TIL, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(e) exposing the second and/or third TIL populations to the transcription factor TF and/or other molecule capable of transiently altering protein expression; the TF and/or other molecule capable of transiently altering protein expression provides an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL population;

(f) harvesting the therapeutic TIL population obtained from step (d); the transition from step (d) to step (e) occurs without opening the system; and

(g) transferring the TIL population harvested from step (e) to an infusion bag; the transition from step (e) to step (f) occurs without opening the system;

(h) optionally, cryopreserving the infusion bag comprising the TIL population harvested from step (f) using a cryopreservation method; and

(i) Administering to the patient a therapeutically effective dose of the third TIL population in the infusion bag of step (g).

45. An expanded TIL population for use in treating a subject having cancer, wherein the expanded TIL population is a third TIL population obtainable by a method comprising:

(a) obtaining a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3 and antigen presenting cell APCs, to produce a third TIL population; performing a second amplification for about 7 to 14 days to obtain a third TIL population; the third TIL population is a therapeutic TIL population; the second amplification is carried out in a closed vessel providing a second gas permeable surface region; the transition from step (c) to step (d) occurs without opening the system;

(e) Exposing the second and/or third TIL populations to the transcription factor TF and/or other molecule capable of transiently altering protein expression; the TF and/or other molecule capable of transiently altering protein expression provides an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL population;

(f) harvesting the therapeutic TIL population obtained from step (d); the transition from step (e) to step (f) occurs without opening the system; and

(g) transferring the TIL population harvested from step (e) to an infusion bag; the transfer of step (f) to step (g) occurs without opening the system; and

(h) optionally, cryopreserving the infusion bag comprising the TIL population harvested from step (f) using a cryopreservation method.

46. The TIL population for use in treating a subject having cancer according to claim 44 or 45, wherein the method further comprises one or more features of any one of claims 1 to 43.

47. A method of expanding a tumor infiltrating lymphocyte TIL into a therapeutic TIL population, wherein the method comprises exposing the TIL to the transcription factor TF and/or other molecule capable of transiently altering protein expression, resulting in a therapeutic TIL population; the TF and/or other molecule capable of transiently altering protein expression provides for increased expression of tumor antigens and/or an increased number of tumor antigen-specific T cells in the therapeutic TIL population.

48. The method of any one of claims 1 to 47, wherein the transient alteration in protein expression results in induction of protein expression.

49. The method of any one of claims 1 to 48, wherein the transient alteration in protein expression results in reduced protein expression.

50. The method of claim 49, wherein more than one sd-RNA is used to reduce transient protein expression.

51. A method for assessing the transcription factor TF and/or other molecules capable of transiently altering the expression of a protein, wherein said method comprises:

expanding the tumor-infiltrating lymphocyte TIL into a therapeutic TIL population, exposing the TIL to transcription factor TF and/or other molecules capable of transiently altering protein expression, producing a therapeutic TIL population; the TF and/or other molecule capable of transiently altering protein expression provides for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population.

52. The method of any one of claims 1 to 51, wherein the transient alteration in protein expression targets a gene selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM3, LAG3, TIGIT, TGF beta, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 alpha), CCL4(MIP 1-beta), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1 and cAMP Protein Kinase A (PKA).

53. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3 to produce a second TIL population; the first amplification is carried out in a closed vessel providing a first gas permeable surface region; performing the first amplification for about 1 to 3 days to obtain a second TIL population; the number of second TIL groups is at least 50 times greater than the number of first TIL groups; the transition from step (c) to step (d) occurs without opening the system;

(e) performing a sterile electroporation step on the second TIL population; the sterile electroporation step mediates transfer of at least one short interfering RNA or one messenger RNA;

(f) standing the second TIL population for about 1 day;

(g) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cell APCs; performing a second amplification for about 7 to 11 days to obtain a third TIL population; the second amplification is carried out in a closed vessel providing a second gas permeable surface region; the transition from step (f) to step (g) occurs without opening the system;

(h) Harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population; the transition from step (g) to step (h) occurs without opening the system; the harvested TIL population is a therapeutic TIL population;

(i) transferring the TIL population harvested from step (h) to an infusion bag; the transfer of step (h) to step (i) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

wherein the step of sterile electroporating comprises delivering a short interfering RNA to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof.

54. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) Performing a sterile electroporation step or an SQZ microfluidic membrane disruption step on the second TIL population; a sterile electroporation step or an SQZ microfluidic membrane disruption step mediates transfer of at least one short interfering RNA or one messenger RNA;

(f) standing the second TIL population for about 1 day;

(h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population; the transition from step (g) to step (h) occurs without opening the system; the harvested TIL population is a therapeutic TIL population;

(i) transferring the TIL population harvested from step (h) to an infusion bag; the transfer of step (h) to step (i) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

wherein the electroporating step comprises delivering a short interfering RNA to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof; and further inserting an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof, into the first TIL group, the second TIL group, or the harvested TIL group by a gamma-retroviral approach or a lentiviral approach.

55. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) performing a sterile electroporation step on the second TIL population; the sterile electroporation step mediates transfer of at least one short interfering RNA or one messenger RNA;

(f) standing the second TIL population for about 1 day;

(i) transferring the TIL population harvested from step (h) to an infusion bag; the transfer of step (h) to step (i) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

wherein the step of sterile electroporation comprises delivering a short interfering RNA to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB, and combinations thereof.

56. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) contacting the first TIL population with at least one sd-RNA; sd-RNA was added at the following concentrations: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium; sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(e) Optionally, performing a sterile electroporation step on the first TIL population; the step of sterile electroporation mediates transfer of at least one sd-RNA;

(f) adding OKT-3 to produce a second TIL population; the first amplification is carried out in a closed vessel providing a first gas permeable surface region; performing the first amplification for about 1 to 3 days to obtain a second TIL population; the number of second TIL groups is at least 50 times greater than the number of first TIL groups; the transition from step (c) to step (f) occurs without opening the system;

(g) standing the second TIL population for about 1 day;

(h) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cell APCs; performing a second amplification for about 7 to 11 days to obtain a third TIL population; the second amplification is carried out in a closed vessel providing a second gas permeable surface region; the transition from step (c) to step (h) occurs without opening the system;

(i) harvesting the therapeutic TIL population obtained from step (h) to provide a harvested TIL population; the transition from step (h) to step (i) occurs without opening the system; the harvested TIL population is a therapeutic TIL population;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; the transfer of step (i) to step (j) occurs without opening the system; and

(k) Harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

57. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) contacting the first TIL population with at least one sd-RNA; sd-RNA was added at the following concentrations: 0.1. mu.M sd-RNA/10,000TIL, 0.5. mu.M sd-RNA/10,000TIL, 0.75. mu.M sd-RNA/10,000TIL, 1. mu.M sd-RNA/10,000TIL, 1.25. mu.M sd-RNA/10,000TIL, 1.5. mu.M sd-RNA/10,000TIL, 2. mu.M sd-RNA/10,000TIL, 5. mu.M sd-RNA/10,000TIL, or 10. mu.M sd-RNA/10,000 TIL; sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(e) optionally, performing a sterile electroporation step on the first TIL population; the step of sterile electroporation mediates transfer of at least one sd-RNA;

(f) Adding OKT-3 to produce a second TIL population; the first amplification is carried out in a closed vessel providing a first gas permeable surface region; performing the first amplification for about 1 to 3 days to obtain a second TIL population; the number of second TIL groups is at least 50 times greater than the number of first TIL groups; the transition from step (c) to step (f) occurs without opening the system;

(g) standing the second TIL population for about 1 day;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; the transfer of step (i) to step (j) occurs without opening the system; and

(k) Harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

58. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) standing the second TIL population for about 1 day;

(f) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and antigen presenting cell APCs; performing a second amplification for about 7 to 11 days to obtain a third TIL population; the second amplification is carried out in a closed vessel providing a second gas permeable surface region; the transition from step (c) to step (f) occurs without opening the system;

(g) Contacting a second population of TILs with at least one sd-RNA during any of step (d), step (e) and/or step (f); sd-RNA was added at the following concentrations: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium; sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(h) optionally, performing a sterile electroporation step on the second TIL population; the step of sterile electroporation mediates transfer of at least one sd-RNA;

(i) harvesting the therapeutic TIL population obtained from step (g) or (h), providing a harvested TIL population; the transition from step (g) to step (i) occurs without opening the system; the harvested TIL population is a therapeutic TIL population;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; the transfer of step (i) to step (j) occurs without opening the system; and

(k) Harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

59. A method of expanding tumor infiltrating lymphocyte TILs into a therapeutic TIL population, wherein the method comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) standing the second TIL population for about 1 day;

(g) Contacting a second population of TILs with at least one sd-RNA during any of step (d), step (e) and/or step (f); sd-RNA was added at the following concentrations: 0.1 μ M sd-RNA/10,000TIL, 0.5 μ M sd-RNA/10,000TIL, 0.75 μ M sd-RNA/10,000TIL, 1 μ M sd-RNA/10,000TIL, 1.25 μ M sd-RNA/10,000TIL, 1.5 μ Msd-RNA/10,000TIL, 2 μ M sd-RNA/10,000TIL, 5 μ M sd-RNA/10,000TIL, or 10 μ M sd-RNA/10,000 TIL; sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(h) optionally, performing a sterile electroporation step on the second TIL population; the step of sterile electroporation mediates transfer of at least one sd-RNA;

(i) harvesting the therapeutic TIL population obtained from step (g) or (h), providing a harvested TIL population; the transition from step (e) to step (h) occurs without opening the system; the harvested TIL population is a therapeutic TIL population;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; the transfer of step (h) to step (i) occurs without opening the system; and

(k) harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

60. The method of any one of claims 56-59, wherein during the first amplification, the sd-RNA is added to the first population of cells twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

61. The method of any one of claims 56-59, wherein during the first amplification, the sd-RNA is added to the second population of cells twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

62. The method of any one of claims 56-61, wherein two sd-RNAs are added to inhibit the expression of two molecules selected from PD-1, LAG-3, TIM-3, CISH and CBLB.

63. The method of any one of claims 56-61, wherein two sd-RNAs are added to inhibit the expression of two molecules selected from:

PD-1 and LAG-3;

PD-1 and TIM-3;

PD-1 and CISH;

PD-1 and CBLB;

LAG-3 and TIM-3;

LAG-3 and CISH;

LAG-3 and CBLB;

TIM-3 and CISH;

TIM-3 and CBLB; and

cish and CBLB.

64. The method of any one of claims 56-62, wherein more than two sd-RNAs are added to inhibit the expression of more than two molecules selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

65. The method of any one of claims 56 to 64, wherein expression of at least one molecule selected from PD-1, LAG-3, TIM-3, CISH and CBLB is reduced by at least 80%, 85%, 90% or 95% in a TIL contacted with at least one sd-RNA.

66. The method of any one of claims 56 to 64, wherein expression of at least one molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB is reduced in TIL contacted with at least one sd-RNA by at least 80%, 85%, 90%, or 95% for at least 12 hours, at least 24 hours, or at least 48 hours.

67. The method of any one of claims 56-64, wherein the sd-RNA is prepared by a method comprising:

in vitro transcription from a linear double-stranded DNA template obtained by polymerase chain reaction, PCR, and suitable for in vitro transcription of the sd-RNA, the linear double-stranded DNA template comprising from 5 'to 3': an RNA polymerase promoter on the coding strand of double-stranded DNA, a 5' untranslated region that is less than 3,000 nucleotides in length and is effective to translate mRNA into a detectable polypeptide upon transfection into a eukaryotic cell, an open reading frame encoding the polypeptide; the polypeptide is heterologous to the cell to be transfected; the polypeptide is selected from: ligands or receptors for immune cells, polypeptides that stimulate or inhibit immune system function, polypeptides that inhibit the function of oncogenic polypeptides, a 3' untranslated region that can efficiently translate mRNA into a detectable polypeptide upon transfection into eukaryotic cells, and a poly (a) stretch of 50 to 5,000 nucleotides on the coding strand of double-stranded DNA; the promoter is heterologous to the open reading frame; the DNA template is not contained within the DNA vector and terminates at the 3' end of the poly (A) stretch.

68. The method of claim 67, wherein the RNA polymerase promoter comprises a consensus binding sequence for an RNA polymerase selected from T7, T3, or SP6 RNA polymerase.

69. The method of claim 67, wherein said open reading frame encodes a polypeptide selected from PD-1, LAG-3, TIM-3, CISH and CBLB.

70. The method of claim 67, wherein the linear double stranded DNA template further comprises an internal ribosomal entry site.

Background

The treatment of large-volume, refractory cancers with adoptive metastatic tumor-infiltrating lymphocytes (TILs) represents an effective approach to the treatment of patients with poor prognosis. Gattinini et al, nat.rev.i mmunol, 2006, 6, 383-one 393. Successful immunotherapy requires large amounts of TIL and commercialization requires robust and reliable methods. This is a challenge to achieve due to technical, logistical and regulatory issues with cell expansion. Due to its speed and efficiency, IL-2 based TIL amplification followed by a "Rapid amplification Process" (REP) has become the preferred method for TIL amplification. Dudley et al, Science, 2002, 298, 850-54; dudley et al, j.clin.oncol., 2005, 23, 2346-57; dudley et al, j.clin.oncol., 2008, 26, 5233-39; riddell et al, Science 1992, 257, 238-41; dudley et al, J.I mmunither, 2003,26, 332-42. REP can expand TIL 1000-fold within 14 days, although it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMC, also known as Monocytes (MNC)) usually from multiple donors as feeder cells, as well as anti-CD 3 antibodies (OKT3) and high doses of IL-2. Dudley et al J.I mmunither.2003, 26,332-42. TILs undergoing the REP program resulted in successful adoptive cell therapy following host immunosuppression in melanoma patients.

There is an urgent need to provide more powerful or effective methods of TIL production and therapies based on such methods that are suitable for commercial scale production and regulatory approval for human patients in multiple clinical centers. The present invention meets this need by providing a transient gene alteration method for reprogramming TILs to produce a therapeutic TIL population with increased therapeutic efficacy.

Disclosure of Invention

The present invention provides improved and/or shortened methods of amplifying TILs and producing therapeutic TIL populations.

The present invention provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising: (i) obtaining a first TIL population from a resected tumor of a patient; (ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population; (iii) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a third TIL population; wherein the number of third TIL groups is at least 100 times greater than the number of second TIL groups; wherein the second amplification is performed for at least 14 days to obtain a third population of TILs; wherein the third TIL population is a therapeutic TIL population; and (iv) exposing the second and/or third TIL populations to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression; wherein TF and/or other molecules capable of transiently altering protein expression provide for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population.

In some embodiments, the method further comprises exposing the second and/or third TIL populations to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression; wherein TF and/or other molecules capable of transiently altering protein expression provide for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population.

The present invention also provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, producing a second TIL population; wherein the first amplification is carried out in a closed vessel providing a first gas permeable surface region; wherein the first amplification is performed for about 3 to 14 days to obtain a second TIL population; wherein the number of second TIL groups is at least 50 times greater than the number of first TIL groups; 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 TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a third TIL population; wherein the second amplification is performed for about 7 to 14 days to obtain a third population of TILs; wherein the third TIL population is a therapeutic TIL population; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) exposing the second and/or third TIL populations to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression; wherein TF and/or other molecule capable of transiently altering protein expression provides for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population;

(f) harvesting the therapeutic TIL population obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system.

In some embodiments, the method further comprises: (iii) performing a further second expansion by supplementing the cell culture medium of the third TIL population with further IL-2, further OKT-3 and further APCs, before or after step (iv); wherein the further second expansion is performed for at least 14 days to obtain a larger population of therapeutic TILs than the population of therapeutic TILs obtained in step (iii); among these, larger therapeutic TIL populations exhibit altered numbers of T cells specific for tumor antigens.

In some embodiments, the method further comprises the steps of: cryopreserving the infusion bag comprising the TIL population harvested from step (f) using a cryopreservation method.

In some embodiments, the cryopreservation method uses a 1: the harvested TIL population at a ratio of 1 was performed on cryopreservation media.

In some embodiments, the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, the PBMCs are irradiated and allogeneic. In some embodiments, the PBMCs are added to the cell culture on any of days 9 to 14 of step (d). In some embodiments, the antigen presenting cell is an artificial antigen presenting cell.

In some embodiments, the harvesting in step (e) is performed using a membrane-based cell processing system.

In some embodiments, the harvesting in step (e) is performed using a LOVO cell processing system.

In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, each fragment having a volume of about 27mm³。

In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, having a total volume of about 1300mm³To about 1500mm³。

In some embodiments, the plurality of fragments comprises about 50 fragments having a total volume of about 1350mm ³。

In some embodiments, the plurality of fragments comprises about 50 fragments and has a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

In some embodiments, the cell culture medium in step (d) further comprises IL-15 and/or IL-21.

In some embodiments, the IL-2 concentration is from about 10,000IU/mL to about 5,000 IU/mL.

In some embodiments, the IL-15 concentration is from about 500IU/mL to about 100 IU/mL.

In some embodiments, the IL-21 concentration is from about 20IU/mL to about 0.5 IU/mL.

In some embodiments, the infusion bag in step (f) is a hypo thermolosol-containing infusion bag.

In some embodiments, the cryopreservation media comprises dimethyl sulfoxide (DMSO). In some embodiments, the cryopreservation media comprises 7% to 10% dimethyl sulfoxide (DMSO).

In some embodiments, the first phase in step (c) and the second phase in step (e) are performed for a period of 10 days, 11 days, or 12 days, respectively.

In some embodiments, the first stage in step (c) and the second stage in step (e) are each performed for a period of 11 days.

In some embodiments, steps (a) through (f) are performed for a period of about 10 days to about 22 days.

In some embodiments, steps (a) through (f) are performed for a period of about 20 days to about 22 days.

In some embodiments, steps (a) through (f) are performed for a period of about 15 days to about 20 days.

In some embodiments, steps (a) through (f) are performed for a period of about 10 days to about 20 days.

In some embodiments, steps (a) through (f) are performed for a period of about 10 days to about 15 days.

In some embodiments, steps (a) through (f) are performed for less than 22 days.

In some embodiments, steps (a) through (f) are performed for less than 20 days.

In some embodiments, steps (a) through (f) are performed for less than 15 days.

In some embodiments, steps (a) through (f) are performed for less than 10 days.

In some embodiments, steps (a) through (f) and cryopreservation are performed for less than 22 days.

In some embodiments, the therapeutic TIL population harvested in step (e) comprises TIL sufficient for a therapeutically effective dose of TIL.

In some embodiments, the amount of TIL sufficient for a therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

In some embodiments, steps (b) through (e) are performed in a single vessel; wherein performing steps (b) through (e) in a single container results in an increased yield of TIL per resected tumor as compared to performing steps (b) through (e) in more than one container.

In some embodiments, during the second phase of step (d), antigen presenting cells are added to the TIL without turning on the system.

In some embodiments, the third TIL population in step (d) provides at least 5-fold or greater interferon-gamma production when administered to a subject.

In some embodiments, the risk of microbial contamination is reduced compared to open systems.

In some embodiments, the TIL from step (f) or step (g) is injected into the patient.

In some embodiments, the plurality of fragments comprises about 4 fragments.

The invention also provides a method of treating a subject having cancer, the method comprising administering expanded Tumor Infiltrating Lymphocytes (TILs), the method comprising:

(a) obtaining a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(e) exposing the second and/or third TIL populations to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression; wherein TF and/or other molecule capable of transiently altering protein expression provides an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL population;

(f) harvesting the therapeutic TIL population obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system;

(h) optionally, cryopreserving the infusion bag comprising the TIL population harvested from step (f) using a cryopreservation method; and

(i) Administering to the patient a therapeutically effective dose of the third TIL population in the infusion bag of step (g).

In some embodiments, the population of therapeutic TILs harvested in step (f) comprises sufficient TILs for administration of a therapeutically effective dose of TILs in step (h).

In some casesIn this embodiment, the amount of TIL sufficient for administering a therapeutically effective dose in step (h) is about 2.3X 10¹⁰To about 13.7X 10¹⁰。

In some embodiments, the Antigen Presenting Cells (APCs) are PBMCs.

In some embodiments, the PBMCs are added to the cell culture on any of days 9 to 14 of step (d).

In some embodiments, the patient has been administered a non-myeloablative (lymphodepletion) regimen prior to administering the therapeutically effective dose of TIL cells in step (h).

In some embodiments, the non-myeloablative lymphocyte depletion regimen comprises a treatment at 60mg/m²Cyclophosphamide was administered at a dose per day for 2 days, then 25mg/m²Dose per day fludarabine was administered for a 5 day period.

In some embodiments, the method further comprises the steps of: beginning treatment of the patient with the high dose IL-2 regimen the next day after administering TIL cells to the patient in step (h).

In some embodiments, the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg every 8 hours as a 15 minute bolus infusion until tolerated.

In some embodiments, the cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer including Head and Neck Squamous Cell Carcinoma (HNSCC), renal cancer, and renal epithelial renal cell carcinoma.

In some embodiments, the cancer is selected from melanoma, HNSCC, cervical cancer, and NSCLC.

In some embodiments, the cancer is melanoma.

In some embodiments, the cancer is HNSCC.

In some embodiments, the cancer is cervical cancer.

In some embodiments, the cancer is NSCLC.

The invention also provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) adding processed tumor fragments from a resected tumor of a patient to a closed system to obtain a first TIL population;

(b) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, producing a second TIL population; wherein the first amplification is carried out in a closed vessel providing a first gas permeable surface region; wherein the first amplification is performed for about 3 to 14 days to obtain a second TIL population; wherein the number of second TIL groups is at least 50 times greater than the number of first TIL groups; 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 of the second TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a third TIL population; wherein the second amplification is performed for about 7 to 14 days to obtain a third population of TILs; wherein the third TIL population is a therapeutic TIL population; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (b) to step (c) occurs without opening the system;

(d) harvesting the therapeutic TIL population obtained from step (c); wherein the transition from step (c) to step (d) occurs without opening the system; and

(e) transferring the TIL population harvested from step (d) to an infusion bag; wherein the transfer of step (d) to step (e) occurs without opening the system.

In some embodiments, the therapeutic TIL population harvested in step (d) comprises TIL sufficient for a therapeutically effective dose of TIL.

In some embodiments, the amount of TIL sufficient for a therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

In some embodiments, the method further comprises the step of cryopreserving the infusion bag comprising the harvested TIL population using a cryopreservation method.

In some embodiments, the cryopreservation method uses a 1: the harvested TIL population at a ratio of 1 was performed on cryopreservation media.

In some embodiments, the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

In some embodiments, the PBMCs are irradiated and allogeneic.

The method of claim 68, wherein the PBMCs are added to the cell culture on any of days 9 to 14 of step (c).

In some embodiments, the antigen presenting cell is an artificial antigen presenting cell.

In some embodiments, the harvesting in step (d) is performed using a LOVO cell processing system.

In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27mm³。

In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, having a total volume of about 1300mm³To about 1500mm³。

In some embodiments, the plurality of fragments comprises about 50 fragments having a total volume of about 1350mm³。

In some embodiments, the plurality of fragments comprises about 50 fragments and has a total mass of about 1 gram to about 1.5 grams.

In some embodiments, the plurality of fragments comprises about 4 fragments.

In some embodiments, the second cell culture medium is provided in a container selected from the group consisting of a G container and a Xuri cell bag.

In some embodiments, the infusion bag in step (e) is a hypo thermolosol-containing infusion bag.

In some embodiments, the first phase in step (b) and the second phase in step (c) are performed for a period of 10 days, 11 days, or 12 days, respectively.

In some embodiments, the first phase in step (b) and the second phase in step (c) are each performed for a period of 11 days.

In some embodiments, steps (a) through (e) are performed for a period of about 10 days to about 22 days.

In some embodiments, steps (a) through (e) are performed for a period of about 10 days to about 20 days.

In some embodiments, steps (a) through (e) are performed for a period of about 10 days to about 15 days.

In some embodiments, steps (a) through (e) are performed for less than 22 days.

In some embodiments, steps (a) through (e) and cryopreservation are performed for less than 22 days.

In some embodiments, antigen presenting cells are added to the TIL during the second stage of step (c) without turning the system on.

In some embodiments, the risk of microbial contamination is reduced compared to open systems.

In some embodiments, the TIL from step (e) is injected into the patient.

In some embodiments, the closed vessel comprises a single bioreactor.

In some embodiments, the closed container comprises G-REX-10.

In some embodiments, the closed container comprises G-REX-100.

In some embodiments, in step (d), the ratio of 25: 1 to 100: APC of 1: TIL ratio Antigen Presenting Cells (APCs) are added to a cell culture of a second TIL population.

In some embodiments, the cell culture has a size of 2.5 × 10⁹APC ratio of 100X 10⁶The ratio of TILs.

In some embodiments, in step (c), the ratio of 25: 1 to 100: APC of 1: TIL ratio Antigen Presenting Cells (APCs) are added to a cell culture of a second TIL population.

In some embodiments, the cell culture has a size of 2.5 × 10⁹APC and 100X 10⁶The ratio of TILs.

The invention also provides an expanded TIL population for use in treating a subject having cancer, wherein the expanded TIL population is a third TIL population obtainable by a method comprising:

(a) obtaining a first TIL population from a tumor resected from a subject by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) Adding tumor fragments to the closed system;

(d) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a third TIL population; wherein the second amplification is performed for about 7 to 14 days to obtain a third population of TILs; wherein the third TIL population is a therapeutic TIL population; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) exposing the second and/or third TIL populations to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression; wherein TF and/or other molecule capable of transiently altering protein expression provides an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL population;

(f) Harvesting the therapeutic TIL population obtained from step (d); wherein the transition from step (d) to step (e) occurs without opening the system; and

(g) transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(h) optionally, cryopreserving the infusion bag comprising the TIL population harvested from step (f) using a cryopreservation method.

In some embodiments, the TIL population is used to treat a subject having cancer according to the methods described above and herein, wherein the method further comprises one or more features described above and herein.

The invention also provides a determination method for determining the activity of the TIL. The present disclosure provides a method of determining Tumor Infiltrating Lymphocytes (TILs) activity by expanding TILs into a larger population of TILs, comprising:

(i) obtaining a first population of previously amplified TILs;

(ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2, producing a second TIL population; and

(iii) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a third TIL population; wherein the number of third TIL groups is at least 100 times greater than the number of second TIL groups; wherein the second amplification is performed for at least 14 days to obtain a third population of TILs; wherein the third TIL population is further analyzed for viability.

In some embodiments, the method further comprises:

(iv) performing an additional second expansion by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3, and additional APCs; wherein a further second amplification is carried out for at least 14 days to obtain a population of TILs that is larger than the population of TILs obtained in step (iii); wherein the third TIL population is further analyzed for viability.

In some embodiments, the cells are cryopreserved prior to step (i).

In some embodiments, the cells are thawed prior to performing step (i).

In some embodiments, step (iv) is repeated 1 to 4 times to obtain sufficient TIL for analysis.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 40 days to about 50 days.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 42 days to about 48 days.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 42 days to about 45 days.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 44 days.

In some embodiments, the cells from step (iii) or (iv) express similar levels of CD4, CD8, and TCR α β as freshly harvested cells.

In some embodiments, the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

In some embodiments, the PBMCs are added to the cell culture on any of days 9 to 17 of step (iii).

In some embodiments, the APC is an artificial APC (aapc).

In some embodiments, the method further comprises the step of transducing the first TIL population with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the method further comprises the step of transducing the first TIL population with an expression vector comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR) comprising a single chain variable fragment antibody fused to at least one intracellular domain (endodomain) of a T cell signaling molecule.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the viability of the TIL is determined.

In some embodiments, the viability of TIL is determined after cryopreservation.

In some embodiments, the viability of the TIL is determined after cryopreservation and after step (iv).

The invention also provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising exposing the TILs to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression, producing a therapeutic TIL population; wherein TF and/or other molecules capable of transiently altering protein expression provide an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the therapeutic TIL population.

In some embodiments, the transient alteration in protein expression results in induction of protein expression.

In some embodiments, the transient alteration in protein expression results in decreased protein expression.

In some embodiments, more than one sd-RNA is used to reduce transient protein expression.

The invention also provides a method of assessing Transcription Factor (TF) and/or other molecules capable of transiently altering protein expression; wherein the method comprises expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, exposing the TILs to Transcription Factors (TFs) and/or other molecules capable of transiently altering protein expression, producing a therapeutic TIL population; wherein TF and/or other molecules capable of transiently altering protein expression provide for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population.

In some embodiments, the transient alteration in protein expression targets a gene selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM3, LAG3, TIGIT, TGF beta, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 alpha), CCL4(MIP 1-beta), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1 and cAMP Protein Kinase A (PKA).

In some embodiments, the present invention provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) contacting the first TIL population with at least one sd-RNA; wherein sd-RNA is added at the following concentrations: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium; wherein sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(e) optionally, performing a sterile electroporation step on the first TIL population; wherein the step of sterile electroporation mediates transfer of at least one sd-RNA;

(f) Adding OKT-3 to produce a second TIL population; wherein the first amplification is carried out in a closed vessel providing a first gas permeable surface region; wherein the first amplification is performed for about 1 to 3 days to obtain a second TIL population; wherein the number of second TIL groups is at least 50 times greater than the number of first TIL groups; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) standing the second TIL population for about 1 day;

(h) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (c) to step (h) occurs without opening the system;

(i) harvesting the therapeutic TIL population obtained from step (h) to provide a harvested TIL population; wherein the transition from step (h) to step (i) occurs without opening the system; wherein the harvested TIL population is a therapeutic TIL population;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; wherein the transfer of step (i) to step (j) occurs without opening the system; and

(k) Harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

In some embodiments, the present invention provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) contacting the first TIL population with at least one sd-RNA, wherein the sd-RNA is added at a concentration of: 0.1. mu.M sd-RNA/10,000TIL, 0.5. mu.M sd-RNA/10,000TIL, 0.75. mu.M sd-RNA/10,000TIL, 1. mu.M sd-RNA/10,000TIL, 1.25. mu.M sd-RNA/10,000TIL, 1.5. mu.M sd-RNA/10,000TIL, 2. mu.M sd-RNA/10,000TIL, 5. mu.M sd-RNA/10,000TIL, or 10. mu.M sd-RNA/10,000 TIL; wherein sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(e) optionally performing a sterile electroporation step on the first TIL population; wherein the step of sterile electroporation mediates transfer of at least one sd-RNA;

(g) standing the second TIL population for about 1 day;

(h) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (g) to step (h) occurs without opening the system;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; wherein the transfer of step (i) to step (j) occurs without opening the system; and

(k) Harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

In some embodiments, the present invention provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) adding OKT-3 to produce a second TIL population; wherein the first amplification is carried out in a closed vessel providing a first gas permeable surface region; wherein the first amplification is performed for about 1 to 3 days to obtain a second TIL population; wherein the number of second TIL groups is at least 50 times greater than the number of first TIL groups; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) standing the second TIL population for about 1 day;

(f) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (c) to step (f) occurs without opening the system;

(g) Contacting a second population of TILs with at least one sd-RNA during any of step (d), step (e) and/or step (f); wherein sd-RNA is added at the following concentrations: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu. Msd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium; wherein sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(h) optionally, performing a sterile electroporation step on the second TIL population; wherein the step of sterile electroporation mediates transfer of at least one sd-RNA;

(i) harvesting the therapeutic TIL population obtained from step (g) or (h), providing a harvested TIL population; wherein the transition from step (g) to step (i) occurs without opening the system; wherein the harvested TIL population is a therapeutic TIL population;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; wherein the transfer of step (i) to step (j) occurs without opening the system; and

(k) Harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

In some embodiments, the present invention provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) standing the second TIL population for about 1 day;

(f) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (e) to step (f) occurs without opening the system;

(g) Contacting a second population of TILs with at least one sd-RNA during any of step (d), step (e) and/or step (f); wherein sd-RNA is added at the following concentrations: 0.1. mu.M sd-RNA/10,000TIL, 0.5. mu.M sd-RNA/10,000TIL, 0.75. mu.M sd-RNA/10,000TIL, 1. mu.M sd-RNA/10,000TIL, 1.25. mu.M sd-RNA/10,000TIL, 1.5. mu.M sd-RNA/10,000TIL, 2. mu.M sd-RNA/10,000TIL, 5. mu.M sd-RNA/10,000TIL, or 10. mu.M sd-RNA/10,000 TIL; wherein sd-RNA is used to inhibit the expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CISH and CBLB and combinations thereof;

(h) optionally, performing a sterile electroporation step on the second TIL population; wherein the step of sterile electroporation mediates transfer of at least one sd-RNA;

(i) harvesting the therapeutic TIL population obtained from step (g) or (h), providing a harvested TIL population; wherein the transition from step (e) to step (h) occurs without opening the system; wherein the harvested TIL population is a therapeutic TIL population;

(j) (ii) transferring the TIL population harvested from step (i) to an infusion bag; wherein the transfer of step (h) to step (i) occurs without opening the system; and

(k) harvested TIL populations were cryopreserved using dimethylsulfoxide-based cryopreservation media.

In some embodiments, sd-RNA is added to the first population of cells twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the first amplification.

In some embodiments, sd-RNA is added to the second population of cells twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the first amplification.

In some embodiments, two sd-RNAs are added to inhibit the expression of two molecules selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

In some embodiments, two sd-RNAs are added to inhibit the expression of two molecules, wherein the two molecules are selected from:

PD-1 and LAG-3;

PD-1 and TIM-3;

PD-1 and CISH;

PD-1 and CBLB;

LAG-3 and TIM-3;

LAG-3 and CISH;

LAG-3 and CBLB;

TIM-3 and CISH;

TIM-3 and CBLB; and

cish and CBLB.

In some embodiments, more than two sd-RNAs are added to inhibit the expression of more than two molecules selected from PD-1, LAG-3, TIM-3, CISH, and CBLB.

In some embodiments, expression of at least one molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB is reduced by at least 80%, 85%, 90%, or 95% in a TIL contacted with at least one sd-RNA.

In some embodiments, expression of at least one molecule selected from PD-1, LAG-3, TIM-3, CISH, and CBLB is reduced by at least 80%, 85%, 90%, or 95% for at least 12 hours, at least 24 hours, or at least 48 hours in a TIL contacted with at least one sd-RNA.

Drawings

FIG. 1: a diagram of an embodiment of process 2A (a 22 day process for producing TIL) is shown.

FIG. 2: a comparison of an embodiment of a 1C process and a 2A process for producing TIL is shown.

FIG. 3: a 1C process timeline is shown.

FIG. 4: the process of an embodiment of TIL therapy (including administration and combination treatment steps) for TIL production using process 2A with a higher cell count is shown.

FIG. 5: the process of an embodiment of TIL therapy (including administration and combination treatment steps) for TIL production using process 2A with a lower cell count is shown.

FIG. 6: a detailed schematic of an embodiment of the 2A process is shown.

Fig. 7A-7C: the main steps of an embodiment of process 2A (including the cryopreservation step) are described.

FIG. 8: an exemplary Process 2A chart, provides an overview of steps A through F.

FIG. 9: process flow diagram for process 2A data collection plan.

FIG. 10: protocol for an exemplary embodiment of a rapid amplification protocol (REP). After tumor arrival, tumors were disrupted and placed in G-Rex flasks containing IL-2 for TIL amplification (pre-REP amplification) for 11 days. For triple mixture studies, IL-2/IL-15/IL-21 was added at the beginning of pre-REP. For the Rapid Expansion Protocol (REP), TIL was cultured with feeder cells and OKT3 for additional 11 days for REP expansion.

FIG. 11: an exemplary production process for TIL stored frozen (about 22 days).

FIG. 12: a diagram of an embodiment of process 2A (a 22 day process for producing TIL) is shown.

FIG. 13: comparative tables for step a through step F of the exemplary embodiments of process 1C and process 2A.

FIG. 14: a detailed comparison of an embodiment of process 1C and an embodiment of process 2A.

FIG. 15: description of an embodiment of the process for the production of cryopreserved TIL (22 days).

FIG. 16: table of process improvements from Gen 1 to Gen 2.

FIG. 17: gen 2 protocol for LN-144 production Process for cryopreservation.

FIG. 18: a diagram of an embodiment of process 2A (a 22 day process for producing TIL) is shown.

FIG. 19: a schematic of the aseptic welding (see procedure note 5.11 of example 16) of the TIL suspension transfer pack to the bottom of a gravity blood filter (single line) is shown.

FIG. 20: a schematic of the sterile welding of the red medium withdrawal line from GRex100MCS (see procedure notes 5.11 of example 16) to the "supernatant" transfer pack is shown.

FIG. 21: a schematic of the welding of 4S-4M60 (see process note 5.11 of example 16) to CC2CellConnect is shown, with the 4 tip (spike) ends of the 4S-4M60 manifold (manifold) replacing the single tips of the CellConnect device (B) at (G).

FIG. 22: a schematic illustration of one of the male luer hub ends of a relay fluid transfer set welded (see procedure note 5.11 of example 16) to 4S-4M60 is shown.

FIG. 23: a schematic of the aseptic welding of the long end of a gravity blood filter (see procedure notes 5.11 of example 16) to a LOVO source bag is shown.

FIG. 24: a schematic of the sterile welding of one of the two source lines of the filter (see procedure note 5.11 of example 16) to a "pooled TIL suspension" collection bag is shown.

FIG. 25: a schematic diagram of the aseptic welding of 4S-4M60 (see procedure note 5.11 of example 16) to CC2CellConnect is shown, with the 4 tip ends of the 4S-4M60 manifold replacing the single tips of the Cell Connect device (B) at (G).

FIG. 26: a schematic representation of the aseptic welding of CS750 cryopreserved bags (see procedure note 5.11 of example 16) to the harness (harness) prepared in step 8.14.8 is shown, replacing one of the 4 male luer connectors (E) with each bag.

FIG. 27 is a schematic view showing: a schematic of a tip for welding a CS-10 pouch (see procedure note 5.11 of example 16) to 4S-4M60 is shown.

FIG. 28: a schematic of the welding of the "formulated til (formulated til)" bag (see process notes 5.11 of example 16) to the remaining tip (a) on the device prepared in step 8.14.10 is shown.

FIG. 29: a schematic of the heat seal at F (see procedure note 5.12 in example 16) is shown with the empty retentate (retenate) and CS-10 bags removed.

FIG. 30: a schematic diagram of an embodiment of the TIL process for transient gene editing is shown.

FIG. 31: a schematic diagram of an embodiment of the TIL process for transient gene editing is shown.

FIG. 32: a schematic diagram is shown for the incorporation of an RNA transfer step into the TIL process for the purpose of transient gene reprogramming.

FIG. 33: an overview of the proposed genetic engineering methods for transiently altering gene expression in TIL is shown.

FIG. 34: a profile of chemokines and chemokine receptors whose transient changes in gene expression are useful for improving TIL trafficking to tumor sites is shown.

FIG. 35: a second profile of chemokines and chemokine receptors is shown, and transient changes in gene expression of these chemokines and chemokine receptors can be used to improve TIL trafficking to tumor sites.

FIG. 36: a schematic structural representation of an exemplary self-delivering ribonucleic acid (sd-RNA) embodiment is shown. See Ligtenberg et al, mol.

FIG. 37: a schematic structural representation of an exemplary sd-RNA embodiment is shown. See U.S. patent publication No. 2016/0304873.

FIG. 38: an exemplary protocol for mRNA synthesis using a DNA template obtained by PCR using specially designed primers is shown. The forward primer contains a phage promoter suitable for in vitro transcription and the reverse primer contains a poly T stretch. The PCR product is an expression cassette (expression cassette) suitable for in vitro transcription. Poly-a at the 3' end of nascent mRNA prevents aberrant RNA uncontrolled (runoff) synthesis and production of double stranded RNA products. After transcription is complete, the polyA tail may be additionally extended with poly (A) polymerase (see U.S. Pat. No. 8,859,229)

FIG. 39: graphs showing Sd-rxRNA-mediated silencing of PDCD1, TIM3, CBLB, LAG3, and CISH.

FIG. 40: Sd-rxRNA mediated gene silencing in TIL; an exemplary scenario. Exemplary tumors include melanoma (fresh or frozen; n ═ 6), breast tumor (fresh or frozen; n ═ 5), lung tumor (n ═ 1), sarcoma (n ═ 1), and/or ovarian cancer (n ═ 1).

FIG. 41: reduced protein expression was detected in 4 of 5 targets. PD 1: n-9, TIM 3: n-8, LAG 3/CISH: n is 2, Cbl-b: n is 2. Preparation from pre-REP melanoma and fresh breast cancer TIL (prep), 2uM sd-rxRNA. Calculated as KD% (100- (100 × (target gene/NTC))).

FIG. 42: Sd-rxRNA-induced KD decreases with time and stimulation. n-3, preparation from pre-REP melanoma TIL, 2uM sd-rxRNA.

FIG. 43: PDCD1 sd-rxRNA slightly affected the activity of TIL. PD1, TIM3 n >6, preparations from pre-REP melanoma/fresh breast cancer TIL. LAG3, CISH n ═ 2, pre-REP melanoma and breast cancer TIL, 2uM sd-rxRNA.

FIG. 44: Sd-rxRNA-mediated KD of PD1 and TIM3 is associated with phenotypic changes (indicative of TIL activation). n-3, preparation from pre-REP melanoma TIL, 2uM sd-rxRNA.

Fig. 45A and 45B: PD1 and TIM3 knocked out by sd-rxRNA did not affect the expression of other suppression/depletion markers. A) And B) n-3, TIM 3: n-2, preparation from pre-REP melanoma TIL, 2uM sd-rxRNA.

FIG. 46: PD1 and TIM3 KD did not significantly improve IFN γ secretion. n-3, preparation from pre-REP melanoma TIL, 2uM sd-rxRNA.

Fig. 47A to 47F: CD107a mobilisation (mobilisation) was not affected by any sd-rxRNA. A) n-6, preparation from pre-REP melanoma TIL, 2uM sd-rxRNA. B) n-2, preparation from pre-REP melanoma and breast cancer TIL, 2uM sd-rxRNA. C) n-3, frozen melanoma and fresh breast cancer TIL. D) n-3, frozen melanoma, and fresh breast and lung cancer TIL. E) And F) n ═ 3, fresh preparations from breast cancer tumors.

FIG. 48: xCELLigence real-time cell analysis (RTCA).

Fig. 49A and 49B: PD1 KD TIL caused greater killing efficiency. A) Representative plot of killing efficiency. B) Representative of n-3, melanoma TIL, 2uM sd-rxRNA.

Fig. 50A and 50B: Sd-rxRNA dose-response (dose-response) experiments. A) n-3, fresh preparation from breast cancer tumor. B) n-3, preparation from pre-REP melanoma TIL.

FIG. 51: Sd-rxRNA mediated CBLB knockdown could not be detected. A) Figure (a). B) Flow cytometry analysis of the figures. n-2, preparations from pre-REP melanoma and fresh breast cancer TIL. Compared to NTC, the mRNA levels of CBLB were unchanged. The protein level of Cbl-b was unchanged as determined using flow cytometry.

Fig. 52A and 52B: the sd-rxRNA mediated gene silencing assay during TIL production in Everest (Iovance) is shown, assessing the TIL phenotype. sd-rxRNA mediated PD-1 knockdown was associated with phenotypic changes (indicative of TIL activation). PD-1, n >6, preparation from pre-REP melanoma/fresh breast cancer TIL, 2uM sd-rxRNA. A) CD25, CCR7, CD27, CD28, CD56, CD95, 4-1BB, and OX 40. B) CD25, CD56, CCR7, 4-1BB and OX 40. N ═ 12, fresh TIL and frozen TIL; breast, melanoma, ovary and lung.

Fig. 53A and 53B: addition of PD1 sd-rxRNA significantly reduced cell growth, but did not reduce TIL viability. A) And (5) performing fold amplification. B) Cell viability. n-7, mammary TIL, sarcoma TIL and lung TIL.

Fig. 54A and 54B: PD1 KD did not improve CD107a mobilization and IFN γ secretion in response to nonspecific stimulation. A) Percentage of CD8 cells expressing CD107a before and after stimulation. B) Secretion of IFN γ before and after stimulation. n-6, melanoma TIL.

Description of sequence listing

SEQ ID NO: 1 is the amino acid sequence of the heavy chain of Moluomamab (muromonab).

SEQ ID NO: 2 is the amino acid sequence of the light chain of molobuzumab.

SEQ ID NO: 3 is the amino acid sequence of recombinant human IL-2 protein.

SEQ ID NO: 4 is the amino acid sequence of aldesleukin.

SEQ ID NO: 5 is the amino acid sequence of the recombinant human IL-4 protein.

SEQ ID NO: 6 is the amino acid sequence of recombinant human IL-7 protein.

SEQ ID NO: 7 is the amino acid sequence of the recombinant human IL-15 protein.

SEQ ID NO: 8 is the amino acid sequence of recombinant human IL-21 protein.

SEQ ID NO: 9 is the amino acid sequence of human 4-1 BB.

SEQ ID NO: 10 is the amino acid sequence of murine 4-1 BB.

SEQ ID NO: 11 is the heavy chain of the 4-1BB agonist monoclonal antibody, urotuzumab (utolimumab) (PF-05082566).

SEQ ID NO: 12 is the light chain of the 4-1BB agonist monoclonal antibody, Utuzumab (PF-05082566).

SEQ ID NO: 13 is the heavy chain variable region (V) of the 4-1BB agonist monoclonal antibody Utuzumab (PF-05082566)_H)。

SEQ ID NO: 14 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody Utuzumab (PF-05082566)_L)。

SEQ ID NO: 15 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody Utosumab (PF-05082566).

SEQ ID NO: 16 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody, Utositumumab (PF-05082566).

SEQ ID NO: 17 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Utosumab (PF-05082566).

SEQ ID NO: 18 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody, Utosumab (PF-05082566).

SEQ ID NO: 19 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody, Utositumumab (PF-05082566).

SEQ ID NO: 20 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody, Utositumumab (PF-05082566).

SEQ ID NO: 21 is the heavy chain of the 4-1BB agonist monoclonal antibody Urelumab (BMS-663513).

SEQ ID NO: 22 is the light chain of the 4-1BB agonist monoclonal antibody Uriluzumab (BMS-663513).

SEQ ID NO: 23 is the heavy chain variable region (V) of 4-1BB agonist monoclonal antibody Uriluzumab (BMS-663513) _H)。

SEQ ID NO: 24 is the light chain variable region (V) of the 4-1BB agonist monoclonal antibody Uriluzumab (BMS-663513)_L)。

SEQ ID NO: 25 is the heavy chain CDR1 of the 4-1BB agonist monoclonal antibody Uriluzumab (BMS-663513).

SEQ ID NO: 26 is the heavy chain CDR2 of the 4-1BB agonist monoclonal antibody Uriluzumab (BMS-663513).

SEQ ID NO: 27 is the heavy chain CDR3 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO: 28 is the light chain CDR1 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO: 29 is the light chain CDR2 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO: 30 is the light chain CDR3 of the 4-1BB agonist monoclonal antibody Uruguzumab (BMS-663513).

SEQ ID NO: 31 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO: 32 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 33 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 34 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 35 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 36 is a linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 37 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 38 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 39 is a linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 40 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 41 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 42 is the Fc domain of the TNFRSF agonist fusion protein.

SEQ ID NO: 43 is a linker of a TNFRSF agonist fusion protein.

SEQ ID NO: 44 is a linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 45 is the linker of the TNFRSF agonist fusion protein.

SEQ ID NO: 46 is the amino acid sequence of 4-1BB ligand (4-1 BBL).

SEQ ID NO: 47 is the soluble portion of the 4-1BBL polypeptide.

SEQ ID NO: 48 is the heavy chain variable region of 4-1BB agonist antibody 4B4-1-1 version 1 (V)_H)。

SEQ ID NO: 49 is the light chain variable region of 4-1BB agonist antibody 4B4-1-1 version 1 (V)_L)。

SEQ ID NO: 50 is the heavy chain variable region of 4-1BB agonist antibody 4B4-1-1 version 2 (V)_H)。

SEQ ID NO: 51 is the light chain variable region of 4-1BB agonist antibody 4B4-1-1 version 2 (V)_L)。

SEQ ID NO: 52 is the heavy chain variable region (V) of 4-1BB agonist antibody H39E3-2_H)。

SEQ ID NO: 53 is the light chain variable region (V) of the 4-1BB agonist antibody H39E3-2_L)。

SEQ ID NO: 54 is the amino acid sequence of human OX 40.

SEQ ID NO: 55 is the amino acid sequence of murine OX 40.

SEQ ID NO: 56 is the heavy chain of the OX40 agonist monoclonal antibody, talilixizumab (MEDI-0562).

SEQ ID NO: 57 is the light chain of the OX40 agonist monoclonal antibody, talizezumab (MEDI-0562).

SEQ ID NO: 58 is the heavy chain variable region (V) of the OX40 agonist monoclonal antibody taliliximab (MEDI-0562)_H)。

SEQ ID NO: 59 is the light chain variable region (V) of the OX40 agonist monoclonal antibody talipexizumab (MEDI-0562)_L)。

SEQ ID NO: 60 is the heavy chain CDR1 of the OX40 agonist monoclonal antibody talizezumab (MEDI-0562).

SEQ ID NO: 61 is the heavy chain CDR2 of the OX40 agonist monoclonal antibody, taliximab (MEDI-0562).

SEQ ID NO: 62 is the heavy chain CDR3 of the OX40 agonist monoclonal antibody talipexizumab (MEDI-0562).

SEQ ID NO: 63 is the light chain CDR1 of the OX40 agonist monoclonal antibody, talizezumab (MEDI-0562).

SEQ ID NO: 64 is the light chain CDR2 of the OX40 agonist monoclonal antibody, talizezumab (MEDI-0562).

SEQ ID NO: 65 is the light chain CDR3 of the OX40 agonist monoclonal antibody, taliximab (MEDI-0562).

SEQ ID NO: 66 is the heavy chain of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 67 is the light chain of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 68 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 11D4_H)。

SEQ ID NO: 69 is the light chain variable region of OX40 agonist monoclonal antibody 11D4 (V)_L)。

SEQ ID NO: 70 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 71 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 72 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 73 is the light chain CDR1 of the OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 74 is the light chain CDR2 of the OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 75 is the light chain CDR3 of OX40 agonist monoclonal antibody 11D 4.

SEQ ID NO: 76 is the heavy chain of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 77 is the light chain of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 78 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 18D8_H)。

SEQ ID NO: 79 light chain variable region (V) of OX40 agonist monoclonal antibody 18D8_L)。

SEQ ID NO: 80 is the heavy chain CDR1 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 81 is the heavy chain CDR2 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 82 is the heavy chain CDR3 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 83 is the light chain CDR1 of the OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 84 is the light chain CDR2 of the OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 85 is the light chain CDR3 of OX40 agonist monoclonal antibody 18D 8.

SEQ ID NO: 86 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody Hu119-122_H)。

SEQ ID NO: 87 is the light chain variable region (V) of OX40 agonist monoclonal antibody Hu119-122_L)。

SEQ ID NO: 88 is the heavy chain CDR1 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO: 89 is the heavy chain CDR2 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO: 90 is the heavy chain CDR3 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO: 91 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO: 92 is the light chain CDR2 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO: 93 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu 119-122.

SEQ ID NO: 94 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody Hu106-222_H)。

SEQ ID NO: 95 is the light chain variable region (V) of OX40 agonist monoclonal antibody Hu106-222_L)。

SEQ ID NO: 96 is the heavy chain CDR1 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO: 97 is the heavy chain CDR2 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO: 98 is the heavy chain CDR3 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO: 99 is the light chain CDR1 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO: 100 is the light chain CDR2 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO: 101 is the light chain CDR3 of OX40 agonist monoclonal antibody Hu 106-222.

SEQ ID NO: 102 is the amino acid sequence of OX40 ligand (OX 40L).

SEQ ID NO: 103 is a soluble portion of an OX40L polypeptide.

SEQ ID NO: 104 is an alternative soluble portion of an OX40L polypeptide.

SEQ ID NO: 105 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 008_H)。

SEQ ID NO: 106 is the light chain variable region (V) of OX40 agonist monoclonal antibody 008_L)。

SEQ ID NO: 107 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 011_H)。

SEQ ID NO: 108 is the light chain variable region (V) of OX40 agonist monoclonal antibody 011_L)。

SEQ ID NO: 109 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 021_H)。

SEQ ID NO: 110 is the light chain variable region (V) of OX40 agonist monoclonal antibody 021_L)。

SEQ ID NO: 111 is the heavy chain variable region (V) of OX40 agonist monoclonal antibody 023_H)。

SEQ ID NO: 112 is the light chain variable region (V) of OX40 agonist monoclonal antibody 023_L)。

SEQ ID NO: 113 is the heavy chain variable region (V) of an OX40 agonist monoclonal antibody _H)。

SEQ ID NO: 114 is the light chain variable region (V) of an OX40 agonist monoclonal antibody_L)。

SEQ ID NO: 115 is the heavy chain variable region (V) of an OX40 agonist monoclonal antibody_H)。

SEQ ID NO: 116 is the light chain variable region (V) of an OX40 agonist monoclonal antibody_L)。

SEQ ID NO: 117 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody_H)。

SEQ ID NO: 118 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody_H)。

SEQ ID NO: 119 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody_L)。

SEQ ID NO: 120 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody_L)。

SEQ ID NO: 121 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody_H)。

SEQ ID NO: 122 is the heavy chain variable region (V) of a humanized OX40 agonist monoclonal antibody_H)。

SEQ ID NO: 123 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody_L)。

SEQ ID NO: 124 is the light chain variable region (V) of a humanized OX40 agonist monoclonal antibody_L)。

SEQ ID NO: 125 is the heavy chain variable region (V) of an OX40 agonist monoclonal antibody_H)。

SEQ ID NO: 126 is the light chain variable region of an OX40 agonist monoclonal antibody(V_L)。

Detailed Description

I. Introduction to the design reside in

Adoptive cell therapy using TIL cultured ex vivo by a Rapid Expansion Protocol (REP) has resulted in successful adoptive cell therapy following host immunosuppression in melanoma patients. Current infusion acceptance parameters depend on the readout of TIL composition (e.g., CD28, CD8, or CD4 positive) as well as the amplification value fold and viability of the REP product.

Current REP protocols rarely take into account the health of the TIL to be injected into a patient. T cells undergo profound metabolic transitions during maturation from naive T cells to effector T cells (see Chang et al, nat. immunol.2016,17,364, herein explicitly incorporated in its entirety, especially for discussion and markers of anaerobic and aerobic metabolism). For example, naive T cells rely on mitochondrial respiration to produce ATP, whereas mature, healthy effector T cells such as TILs are highly glycolytic, relying on aerobic glycolysis to provide the bioenergetic substrates required for their proliferation, migration, activation and antitumor efficacy.

The current TIL production process is limited by length, cost, sterility issues, and other factors described herein, such that the potential for commercialization of such methods is severely limited; for these and other reasons, no commercial process is currently available. The present invention provides TIL manufacturing methods and therapies based on such methods that utilize transient protein expression alteration methods that are suitable for commercial scale production and regulatory approval for human patients for multiple clinical centers. The present invention provides transient gene alteration methods for reprogramming TILs to produce therapeutic TIL populations with increased therapeutic efficacy.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications cited herein are incorporated by reference in their entirety.

The term "in vivo" refers to an event that occurs in a subject.

The term "in vitro" refers to an event that occurs outside of the body of a subject. In vitro assays include cell-based assays using live or dead cells, and may also include cell-free assays that do not use whole cells.

The term "ex vivo" refers to an event involving the treatment or procedure of a cell, tissue and/or organ that has been removed from the body of a subject. Suitably, the cell, tissue and/or organ may be returned to the subject during surgery or a method of treatment.

The term "rapid expansion" refers to an increase in the amount of antigen-specific TIL of at least about 3 fold (or 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, or 9 fold) over a period of one week, more preferably at least about 10 fold (or 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, or 90 fold) over a period of one week, or most preferably at least about 100 fold over a period of one week. Some rapid amplification protocols are summarized below.

As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to a population of cells originally obtained as leukocytes that have left the subject's bloodstream and migrated to the tumor. TILs include, but are not limited to, CD8⁺Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4⁺T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. "Primary TILs" are those obtained from a patient tissue sample as described herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell population that has been expanded or propagated as discussed herein, including but not limited to a plurality of TILs (bulkTILs) and expanded TILs ("REP TILs" or "post-REP TILs"). The TIL cell population may include genetically modified TILs.

Herein, a "cell population" (including TILs) refers to several cells having common characteristics. In general, the number of clusters is usually 1X 10⁶To 1X 10¹⁰Different TIL groups contain different numbers. For example, in the presence of IL-2, an initial increase in primary TIL yields approximately 1X 10⁸A large TIL population of individual cells. Typically, REP amplification is performed to provide 1.5X 10⁹To 1.5X 10¹⁰Individual cell populations were used for infusion.

"cryopreserved TIL" herein refers to primary, bulk or expanded TIL (REP TIL) processed and stored at about-150 ℃ to-60 ℃. General methods for cryopreservation are also described elsewhere herein, including in the examples. For clarity, "cryopreserved TILs" can be distinguished from frozen tissue samples that can be used as a source of primary TILs.

As used herein, "thawed cryopreserved TILs" refers to TIL populations that have been previously cryopreserved and then processed to return to a temperature above room temperature (including but not limited to cell culture temperature or a temperature at which TILs can be administered to a patient).

The term "cryopreservation medium" or "cryopreservation medium" refers to any medium that can be used for cryopreservation of cells. Such media may include media comprising 7% to 10% DMSO. Exemplary media include CryoStor CS10, hyperthermosol, and combinations thereof. The term "CS 10" refers to cryopreservation media commercially available from Stemcell Technologies or Biolife Solutions. CS10 Medium is available under the trade name " CS10 ". CS10 medium is serum-free, animal component-free medium containing DMSO.

The term "central memory T cell" refers to CD45R0+ in human cells and constitutively expresses CCR7(CCR 7)^{Height of}) And CD62L (CD 62)^{Height of}) A subpopulation of T cells. The surface phenotypes of central memory T cells also include TCR, CD3, CD127(IL-7R) and IL-15R. Transcription factors for central memory T cells include BCL-6, BCL-6B, MBD2 and BMI 1. At TCR contactAfter this time, central memory T cells secrete mainly IL-2 and CD40L as effector molecules. Central memory T cells predominate in the CD4 compartment (component) in the blood and are ratiometrically enriched in lymph nodes and tonsils in humans.

The term "effector memory T cells" refers to a subset of human or mammalian T cells that, like central memory T cells, are CD45R0+, but lose constitutive expression of CCR7(CCR 7)^{Is low in}) And low heterogeneity or expression of CD62L (CD 62L)^{Is low in}). The surface phenotypes of central memory T cells also include TCR, CD3, CD127(IL-7R) and IL-15R. Transcription factors for central memory T cells include BLIMP 1. Effector memory T cells rapidly secrete high levels of inflammatory cytokines including interferon-gamma, IL-4 and IL-5 following antigen stimulation. Effector memory T cells predominate in the CD8 compartment of the blood and are ratioed enriched in the lungs, liver and intestine in humans. CD8 ⁺Effector memory T cells carry large amounts of perforin.

The term "closed system" refers to a system that is closed to the external environment. Any closed system suitable for cell culture processes can be used in the methods of the invention. Closed systems include, for example, but are not limited to, closed G-containers. Once the tumor fragments are added to the closed system, the system is not opened to the external environment until the TIL is ready for administration to the patient.

The terms "fragmenting", "fragmenting" and "fragmented" as used herein to describe methods of disrupting a tumor include mechanical disruption methods such as, for example, fragmenting, slicing, segmenting and comminuting tumor tissue, as well as any other method of disrupting the physical structure of tumor tissue.

The terms "peripheral blood mononuclear cells" and "PBMCs" refer to peripheral blood cells with a circular nucleus, including lymphocytes (T cells, B cells, NK cells) and monocytes. Preferably, the peripheral blood mononuclear cells are irradiated allogeneic peripheral blood mononuclear cells. PBMC is an antigen presenting cell.

The term "anti-CD 3 antibody" refers to an antibody or variant thereof (e.g., a monoclonal antibody), including: a human, humanized, chimeric or murine antibody directed against the CD3 receptor in the T cell antigen receptor of mature T cells. anti-CD 3 antibodies include OKT-3, also known as molobuzumab. anti-CD 3 antibodies also include the UHCT1 clone, also known as T3 and CD 3. Other anti-CD 3 antibodies include, for example, ottelizumab (otelixizumab), teleprizumab (teplizumab), and wilsonizumab (visilizumab).

The term "OKT-3" (also referred to herein as "OKT 3") refers to a monoclonal antibody or biological analog or variant thereof, including human, humanized, chimeric, or murine antibodies directed against CD3 receptor in T cell antigen receptors of mature T cells, and also including commercially available forms such as OKT-3(30ng/mL, pure MACS GMP CD3, Miltenyi Biotech, inc., san diego, ca, usa) and molobroma or variants, conservative amino acid substitutions, glycoforms (glycoforms) or biological analogs thereof. The amino acid sequences of the heavy and light chains of Moluomamab are given in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Hybridomas capable of producing OKT-3 are deposited at the American Type culture Collection (ATCC, American Type culture Collection) and assigned ATCC accession number CRL 8001. Hybridomas capable of producing OKT-3 are also deposited at the European Collection of certified cell cultures (ECACC) and are assigned catalog number 86022706.

Table 1: amino acid sequence of Moluomamab

The term "IL-2" (also referred to herein as "IL 2") refers to a T cell growth factor known as interleukin-2, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs, and variants thereof. IL-2 is described, for example, in Nelson, J.I mmunol.2004,172,3983-88 and Malek, Annu.Rev.I mmunol.2008,26,453-79, the disclosures of which are incorporated herein by reference. The amino acid sequence of recombinant human IL-2 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 3). For example, the term IL-2 includes human recombinant forms of IL-2, e.g., aldesleukin (PROLEUKIN, commercially available from multiple suppliers, 2200 million IU in each vial used individually), as well as recombinant forms of IL-2 commercially available from CellGenix, Inc. (CELLGRO GMP), of CellGen, Inc., of CellHitson, N.H., or ProSpec-Tany TechnoGene Ltd. (catalog number CYT-209-b), of east Brownian, N.H., and other commercial equivalents of other suppliers. Aldesleukin (des-alkyl-1, serine-125 human IL-2) is a non-glycosylated human recombinant form of IL-2 with a molecular weight of about 15 kDa. The amino acid sequence of aldesleukin suitable for use in the present invention is given in Table 2 (SEQ ID NO: 4). The term IL-2 also includes pegylated forms of IL-2 as described herein, including pegylated IL2 prodrug NKTR-214, commercially available from Nektar Therapeutics, inc. NKTR-214 and pegylated IL-2 suitable for use in the present invention are described in U.S. patent application publication No. US2014/0328791A1 and International patent application publication No. WO2012/065086A1, the disclosures of which are incorporated herein by reference. Alternative forms of conjugated IL-2 suitable for use in the present invention are described in U.S. Pat. nos. 4,766,106, 5,206,344, 5,089,261 and 4,902,502, the disclosures of which are incorporated herein by reference. IL-2 formulations suitable for use in the present invention are described in U.S. Pat. No. 6,706,289, the disclosure of which is incorporated herein by reference.

Table 2: amino acid sequence of interleukin

The term "IL-4" (also referred to herein as "IL 4") refers to a cytokine known as interleukin 4, which is produced by Th 2T cells and eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (Th0 cells) into Th 2T cells. Steinke and Borish, respir. res.2001,2, 66-70. Th 2T cells subsequently produce additional IL-4 in a positive feedback loop after activation by IL-4. IL-4 also stimulates B cell proliferation and MHC class II expression and induces B cell switching to IgE and IgG1 expression. Recombinant human IL-4 suitable for use in the present invention is commercially available from a variety of suppliers, including ProSpec-Tany TechnoGene Ltd, N.J. (Cat. CYT-211) and ThermoFisher Scientific, Inc., of Waltham, MA, U.S.A. (human IL-15 recombinant protein, Cat. Gibco CTP 0043). The amino acid sequence of recombinant human IL-4 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 5).

The term "IL-7" (also referred to herein as "IL 7") refers to a glycosylated tissue-derived cytokine called interleukin 7, which is obtainable from stromal and epithelial cells as well as dendritic cells. Fry and Mackall, Blood 2002,99, 3892-904. IL-7 can stimulate the growth of T cells. IL-7 binds to the IL-7 receptor, which is a heterodimer consisting of the IL-7 receptor alpha and a common gamma chain receptor (common gamma chain receptor) that plays an important role in the intraphymotic development and peripheral survival of T cells in a range of signals. Recombinant human IL-7 suitable for use in the present invention is commercially available from a variety of suppliers, including ProSpec-Tany technoGene Ltd, east Brownian Vickers, N.J. (Cat. No. CYT-254) and ThermoFisher Scientific, Inc., of Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. GibcoPHC 0071). The amino acid sequence of recombinant human IL-7 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 6).

The term "IL-15" (also referred to herein as "IL 15") refers to a T cell growth factor known as interleukin-15, and includes all forms of IL-2, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs, and variants thereof. IL-15 is described, for example, in Fehniger and Caligiuri, Blood 2001,97,14-32, the disclosures of which are incorporated herein by reference. IL-15 shares beta and gamma signaling receptor subunits with IL-2. Recombinant human IL-15 is a single non-glycosylated polypeptide chain, containing 114 amino acids (and an N-terminal methionine), and having a molecular weight of 12.8 kDa. Recombinant human IL-15 is commercially available from a variety of suppliers, including ProSpec-Tany technoGene Ltd, east Bronsted, N.J. (Cat. No. CYT-230-b) and ThermoFisher Scientific, Inc., of Waltham, MA, USA (human IL-15 recombinant protein, Cat. No. 34-8159-82). The amino acid sequence of recombinant human IL-15 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 7).

The term "IL-21" (also referred to herein as "IL 21") refers to a pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21, including human and mammalian forms, conservative amino acid substitutions, glycoforms, biological analogs, and variants thereof. IL-21 is described, for example, in Spolski and Leonard, nat. Rev. drug. disc.2014,13,379-95, the disclosures of which are incorporated herein by reference. IL-21 is composed primarily of natural killer T cells and activated human CD4 ⁺T cell production. The recombinant human IL-21 is a non-glycosylated polypeptide single chain, contains 132 amino acids, and has a molecular weight of 15.4 kDa. Recombinant human IL-21 is commercially available from a variety of suppliers, including ProSpec-Tany technoGene Ltd, east Bronsted, N.J. (Cat. No. CYT-408-b) and ThermoFisher Scientific, Inc., of Waltham, MA, USA (human IL-21 recombinant protein, Cat. No. 14-8219-80). The amino acid sequence of recombinant human IL-21 suitable for use in the present invention is given in Table 2 (SEQ ID NO: 8).

When referring to an "anti-tumor effective amount", "tumor inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and patient (subject) condition. In general, it can be said that a pharmaceutical composition comprising a tumor infiltrating lymphocyte (e.g., a secondary TIL or a genetically modified cytotoxic lymphocyte) as described herein can be 10⁴To 10¹¹One cell/kg body weight (e.g., 10)⁵To 10⁶、10⁵To 10¹⁰、10⁵To 10¹¹、10⁶To 10¹⁰、10⁶To 10¹¹、10⁷To 10¹¹、10⁷To 10¹⁰、10⁸To 10¹¹、10⁸To 10¹⁰、10⁹To 10¹¹Or 10⁹To 10¹⁰Individual cells/kg body weight), including all integer values within these ranges. Compositions of tumor infiltrating lymphocytes (in some cases, including genetically modified cytotoxic lymphocytes) can also be administered multiple times at these doses. Tumor infiltrating lymphocytes (in some cases, including genetically) can be treated by using immunization Commonly known infusion techniques are used in therapy (see, e.g., Rosenberg et al, "New" Eng.J.of Med.319: 1676,1988). By monitoring the patient for signs of disease and adjusting the treatment accordingly, one skilled in the medical arts can readily determine the optimal dosage and treatment regimen for a particular patient.

The term "hematologic malignancy" refers to mammalian cancers and tumors of hematopoietic and lymphoid tissues, including but not limited to blood, bone marrow, lymph nodes, and tissues of the lymphatic system. Hematological malignancies are also known as "liquid tumors". Hematologic malignancies include, but are not limited to, Acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Lymphoma (CLL), Small Lymphocytic Lymphoma (SLL), acute myeloid leukemia (a ML), Chronic Myelogenous Leukemia (CML), acute monocytic leukemia (AMoL), hodgkin lymphoma and non-hodgkin lymphoma. The term "B cell hematologic malignancy" refers to a hematologic malignancy that affects B cells.

The term "solid tumor" refers to an abnormal tissue mass that generally does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term solid tumor cancer refers to malignant, neoplastic or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, malignant epithelial tumors, and lymphomas, such as lung, breast, prostate, colon, rectal, and bladder cancers. The tissue structure of a solid tumor comprises interdependent tissue compartments including parenchymal (cancer cells) and supporting stromal cells (into which cancer cells are dispersed and which may provide a supporting microenvironment).

The term "liquid tumor" refers to an abnormal mass of cells that is fluid in nature. Liquid tumor cancers include, but are not limited to, leukemia, myeloma, and lymphoma, as well as other hematological malignancies. TILs obtained from liquid tumors may also be referred to herein as Marrow Infiltrating Lymphocytes (MILs).

As used herein, the term "microenvironment" may refer to the entire solid or hematologic tumor microenvironment, or to a single subpopulation of cells within the microenvironment. As used herein, a tumor microenvironment refers to a complex mixture of "cells, soluble factors, signaling molecules, extracellular matrix, and mechanistic cues that promote tumor transformation, support tumor growth and invasion, protect tumors from host immunity, promote resistance to therapy, and provide a niche for the propagation of dominant metastases," as described in Swartz et al, Cancer res, 2012, 72, 2473. Although tumors express antigens that should be recognized by T cells, the immune system rarely clears tumors due to immunosuppression of the microenvironment.

In one embodiment, the invention includes a method of treating cancer with a TIL population; wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of the TIL of the invention. In some embodiments, a TIL population may be provided; wherein the patient is pre-treated with non-myeloablative chemotherapy prior to infusion of a TIL of the invention. In one embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg/kg/day for 2 days (27 and 26 days before TIL infusion) and fludarabine 25mg/m ²Day/day for 5 days (27 th to 23 th day before TIL infusion). In one embodiment, following non-myeloablative chemotherapy and TIL infusion (on day 0) according to the invention, the patient receives an intravenous infusion of IL-2 at 720,000IU/kg every 8 hours until physiologically tolerated.

Experimental findings indicate that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in enhancing therapeutic efficacy by eliminating the competing components of the regulatory T cells and the immune system ("cytokine deposition"). Thus, some embodiments of the invention employ a lymphocyte depletion step (sometimes also referred to as "immunosuppression modulation") on the patient prior to introducing the rtils of the invention.

As used herein, the terms "co-administration", "administration in combination with.. or", "simultaneous", and "co-administration" include the administration of two or more active pharmaceutical ingredients (in a preferred embodiment of the invention, e.g., at least one potassium channel agonist in combination with a multi-TIL population) to a subject such that the active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time. Co-administration includes: the different compositions are administered simultaneously, at different times, or in the presence of two or more active pharmaceutical ingredients. It is preferred to administer the different compositions simultaneously and to administer the composition in which both agents are present.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or combination of compounds as described herein sufficient to effect the intended use, including but not limited to disease treatment. The therapeutically effective amount may vary depending on the intended use (in vitro or in vivo) or the subject and disease condition being treated (e.g., weight, age and sex of the subject), the severity of the disease condition, or the mode of administration. The term also applies to doses that will induce a specific response in the target cells, such as a reduction in platelet adhesion and/or cell migration. The specific dosage will depend upon the particular compound selected, the administration regimen to be followed, whether the compound is administered in combination with other compounds, the time of administration, the tissue of administration, and the physical delivery system carrying the compound.

The terms "treatment", "treating", and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or therapeutic in terms of a partial or complete cure for the disease and/or adverse effects caused by the disease. As used herein, "treatment" includes any treatment of a disease in a mammal, particularly a human, including: (a) preventing disease development in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting a disease, i.e., arresting its development or progression; and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more symptoms of the disease. "treating" is also meant to include delivering an agent to provide a pharmacological effect, even in the absence of a disease or condition. For example, "treating" includes delivering a composition that can elicit an immune response or confer immunity in the absence of disease, such as in the case of a vaccine.

The term "heterologous" when used in reference to a nucleic acid or protein moiety means that the nucleic acid or protein comprises two or more subsequences that are not in the same relationship to each other in nature. For example, nucleic acids are often recombinantly produced, having more than two sequences from unrelated genes, arranged to produce new functional nucleic acids, such as a promoter from one source and a coding region from another source, or coding regions from different sources. Similarly, a heterologous protein means that the protein comprises two or more subsequences that are not in the same relationship to each other in nature (e.g., a fusion protein).

The terms "sequence identity", "percent identity", and "percent sequence identity" (or synonyms thereof, such as "99% identical") in the context of two or more nucleic acids or polypeptides refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, without regard to any conservative amino acid substitutions as part of sequence identity. Percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art for obtaining alignments of amino acid or nucleotide sequences. Suitable programs for determining percent sequence identity include, for example, the BLAST suite of programs available from the national center for Biotechnology information BLAST website of the U.S. government. The comparison between two sequences can be performed using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. ALIGN, ALIGN-2 (Genentech, south san Francisco, Calif.) or MegAlign, available from DNASTAR, are other publicly available software programs that can be used to ALIGN sequences. One skilled in the art can determine the appropriate parameters for maximum alignment by the particular alignment software. In some embodiments, the alignment software default parameters are used.

As used herein, the term "variant" includes, but is not limited to, an antibody or fusion protein as follows: which comprises an amino acid sequence that differs from the amino acid sequence of the reference antibody by one or more substitutions, deletions and/or additions at certain positions within or near the amino acid sequence of the reference antibody. A variant may comprise more than one conservative substitution in its amino acid sequence compared to the amino acid sequence of a reference antibody. Conservative substitutions may involve, for example, substitution of similarly charged or uncharged amino acids. The variant retains the ability to specifically bind to the antigen of the reference antibody. The term "variant" also includes pegylated antibodies or proteins.

As used herein, "tumor infiltrating lymphocytes" or "TILs" refer to a population of cells originally obtained as leukocytes that have left the subject's bloodstream and migrated to the tumor. TILs include, but are not limited to, CD8⁺Cytotoxic T cells (lymphocytes), Th1 and Th17 CD4⁺T cells, natural killer cells, dendritic cells, and M1 macrophages. TILs include primary TILs and secondary TILs. "primary TILs" are those obtained from patient tissue samples as described herein (sometimes referred to as "freshly harvested"), and "secondary TILs" are any TIL cell population that has been expanded or propagated as discussed herein, including but not limited to bulk TILs, expanded TILs ("REP TILs"), and "REP TILs". For example, the rep TIL may include a second amplification of the TIL or an additional second amplification of the TIL (e.g., those described in step D of fig. 8, including TILs referred to as rep TILs).

TILs can be defined biochemically using cell surface markers, or functionally by their ability to infiltrate a tumor and affect therapy. TILs can be generally classified by expressing more than one of the following biomarkers: CD4, CD8, TCR α β, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD 25. In addition, or alternatively, the TIL may be functionally defined by its ability to infiltrate a solid tumor after its reintroduction into the patient. TIL may also be characterized by potency-for example, a TIL may be considered effective if, for example, the release of Interferon (IFN) is greater than about 50pg/mL, greater than about 100pg/mL, greater than about 150pg/mL, or greater than about 200 pg/mL.

The term "deoxyribonucleotides" encompasses both natural and synthetic unmodified and modified deoxyribonucleotides. Modifications include changes in the linkages between sugar moieties, base moieties and/or deoxyribonucleotides in an oligonucleotide.

The term "RNA" defines a molecule comprising at least one ribonucleotide residue. The term "ribonucleotide" defines a nucleotide having a hydroxyl group at the 2' position of the b-D-ribofuranose moiety. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and altered RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of more than one nucleotide). The nucleotides of the RNA molecules described herein can also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs.

The term "modified nucleotide" refers to a nucleotide that has one or more modifications to the nucleoside, nucleobase, pentose ring or phosphate group. For example, modified nucleotides do not include ribonucleotides that contain adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate, and deoxyribonucleotides that contain deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include modifications that occur naturally as a result of modification of the enzyme that modifies the nucleotide (e.g., methyltransferase).

Modified nucleotides also include synthetic nucleotides or non-naturally occurring nucleotides. Synthetic modifications or non-naturally occurring modifications in nucleotides include modifications having a 2 'modification, (e.g., 2' -O-methyl, 2 '-methoxyethoxy, 2' -fluoro, 2 '-allyl, 2' -O- [2- (methylamino) -2-oxyethyl]4 '-thio, 4' -CH₂-O-2 '-bridge, 4' - (CH)₂)₂-O-2' -bridge, 2' -LNA and 2' -O- (N-methyl carbamate)) or those comprising base analogues. With respect to 2 '-modified nucleotides as described in the present disclosure, "amino" refers to 2' -NH, modified or unmodified₂Or 2' -O- -NH₂. For example, such modifying groups are described in U.S. Pat. nos. 5,672,695 and 6,248,878; incorporated herein by reference.

The term "microRNA" or "miRNA" refers to a nucleic acid that forms a single-stranded RNA that has the ability to alter the expression of a gene or target gene (target gene) (reduce or inhibit expression; modulate expression; directly or indirectly enhance expression) when the miRNA is expressed in the same cell. In one embodiment, miRNA refers to a nucleic acid that is substantially or completely identical to a target gene and forms a single-stranded miRNA. In some embodiments, the miRNA may be in the form of a pre-miRNA, wherein the pre-miRNA is a double-stranded RNA. The sequence of the miRNA may correspond to the full-length target gene or a subsequence thereof. Typically, mirnas are at least about 15 to 50 nucleotides in length (e.g., single-stranded mirnas each sequence is 15 to 50 nucleotides in length, double-stranded pre-mirnas are about 15 to 50 base pairs in length). In some embodiments, the miRNA is 20 to 30 base nucleotides. In some embodiments, the miRNA is 20 to 25 nucleotides in length. In some embodiments, the miRNA is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

The term "target gene" includes genes known or identified to modulate the expression of genes involved in immune resistance mechanisms, which may be one of several groups of genes, for example, inhibitory receptors such as CTLA4 and PD 1; cytokine receptors that inactivate immune cells, such as TGF- β receptor, LAG3, and/or TIM3, and combinations thereof. In some embodiments, the target gene comprises one or more of PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric costimulatory receptor), IL-2, IL-4, IL-7, IL-10, IL-12, IL-15, IL-21, NOTCH 1/2 intracellular domain (ICD), intercellular domain, NOTCH ligand mDLL1, TIM3, LAG3, TIGIT, TGF β, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP kinase.

The term "small interfering RNA" or "siRNA" or "short interfering RNA" or "silencing RNA" defines a set of double-stranded RNA molecules, including a sense RNA strand and an antisense RNA strand, each strand typically being about 1022 nucleotides in length, optionally containing a 3' overhang (overhang) of 1 to 3 nucleotides. sirnas are active in the RNA interference (RNAi) pathway and interfere with the expression of a particular target gene through complementary nucleotide sequences.

The term sd-RNA refers to a "self-delivering" RNAi agent that becomes an asymmetric double-stranded RNA-antisense oligonucleotide hybrid. Double-stranded RNA includes a guide (sense) strand of about 19 to 25 nucleotides and a follower (antisense) strand of about 10 to 19 nucleotides, and forms a duplex, resulting in a single-stranded phosphorothioated tail of about 5 to 9 nucleotides. In some embodiments, RNA sequences can be modified with stabilizing and hydrophobic modifications (e.g., sterols, such as cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and phenyl) that provide stability and efficient cellular uptake in the absence of any transfection reagent or agent. In some embodiments, the immunoassay detection of an IFN-induced protein indicates that sd-RNA produces reduced immunostimulatory activity compared to other RNAi agents. See, e.g., Byrne et al, 12 months 2013, J. ocular Pharmacology and Therapeutics, 29 (10): 855, 864, which is incorporated herein by reference. In some embodiments, sd-RNA described herein is available from Advirna LLC (Worcester, MA, USA).

The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Other active pharmaceutical ingredients (e.g., other drugs) may also be incorporated into the described compositions and methods.

The terms "about" and "approximately" mean within a statistically significant range of values. Such a range may be within an order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, more preferably within 10%, even more preferably within 5%. The permissible variations encompassed by the terms "about" or "approximately" depend on the particular system under study and can be readily understood by one of ordinary skill in the art. Further, as used herein, the terms "about" and "approximately" mean that the size, dimensions, formulation, parameters, shape, and other properties and characteristics are not, and need not be, exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, as well as other factors known to those of skill in the art. Generally, the size, dimension, formulation, parameter, shape or other property or characteristic is "about" or "approximately" whether or not explicitly stated. It should be noted that embodiments having very different sizes, shapes and sizes may employ the described arrangement.

The transitional terms "comprising," "consisting essentially of, and" consisting of, when used in the appended claims in both original and modified form, define the claim scope with respect to additional claim elements or steps (if any) not listed that are excluded from the claim scope. The term "comprising" is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step, or material. The term "consists of" does not include any elements, steps or materials other than those recited in the claims, and in the case of materials, the term "consists of" does not include the usual impurities associated with the specified material. The term "consisting essentially of" limits the scope of the claims to the specified elements, steps or materials and those that do not materially affect the basic and novel characteristics of the claimed invention. In alternative embodiments, all of the compositions, methods, and kits embodying the present invention described herein may be more specifically defined by any of the transitional terms "comprising," "consisting essentially of, and" consisting of.

Method for transiently altering protein expression in TIL

In some embodiments, the amplified TILs of the invention are further manipulated to alter protein expression before, during, or after the amplification step (including during the closed sterile production method as described herein). In some embodiments, the transiently altered protein expression is due to transient gene editing. In some embodiments, the amplified TILs of the invention are treated with Transcription Factors (TFs) and/or other molecules capable of transiently altering protein expression in the TILs. In some embodiments, TF and/or other molecule capable of transiently altering protein expression provides for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the TIL population.

In some embodiments, the invention includes gene editing by nucleotide insertion (e.g., by ribonucleic acid (RNA) insertion, including messenger RNA (mrna) or small (or short) interfering RNA (sirna) insertion) TIL populations to promote expression of or inhibit expression of more than one protein, and simultaneously promote the combination of one set of proteins with the inhibition of another set of proteins.

In some embodiments, the amplified TILs of the invention undergo transient changes in protein expression. In some embodiments, the transient change in protein expression occurs in a plurality of TIL populations prior to the first amplification, including, for example, the TIL population obtained by, for example, step a shown in fig. 8. In some embodiments, the transient change in protein expression occurs during the first amplification, including a population of TILs amplified, e.g., in step B, such as shown in fig. 8. In some embodiments, the transient change in protein expression occurs after the first amplification, the TIL population comprising, e.g., the transition between the first amplification and the second amplification, the TIL population obtained, e.g., in step B and included in step C, as shown in fig. 8. In some embodiments, the transient change in protein expression occurs in a plurality of TIL populations prior to the second amplification, including TIL populations obtained, for example, by step C and prior to the amplification of step D, as shown, for example, in fig. 8. In some embodiments, the transient change in protein expression occurs during a second amplification, including a population of TILs amplified, e.g., in step D, as shown, e.g., in fig. 8. In some embodiments, the transient alteration in protein expression occurs after a second amplification, including, for example, the TIL population obtained from the amplification, e.g., step D, shown in fig. 8.

In one embodiment, the method of transiently altering protein expression in a TIL population comprises the step of electroporation. In one embodiment, the method for transiently altering the expression of a protein in a TIL population is performed according to the method shown in fig. 30 and 31. Electroporation methods are known in the art and are described, for example, in Tsong, biophysis.j.1991, 60, 297-306 and U.S. patent application publication No. 2014/0227237a1, the respective disclosures of which are incorporated herein by reference. In one embodiment, the method for transiently altering protein expression in a TIL population comprises the step of calcium phosphate transfection. Calcium phosphate transfection methods (calcium phosphate DNA precipitation, cell surface coating and endocytosis) are known in the art and are described in Graham and van der Eb, Virology1973, 52, 456-467; wigler et al, Proc.Natl.Acad.Sci.1979, 76, 1373-1376; chen and Okayarea, mol.cell.biol.1987, 7, 2745-; and U.S. patent No. 5,593,875, the disclosure of each of which is incorporated herein by reference. In one embodiment, the method for transiently altering protein expression in a TIL population comprises the step of lipofection. Lipofectin methods, for example, 1: methods for 1(w/w) lipid formulations are known in the art and are described in Rose et al, Biotechniques 1991, 10, 520-525 and Felgner et al, Proc. Natl. Acad. Sci. USA, 1987, 84, 7413-7417 and US patent No. 5279833; 5,908,635, respectively; 6,056,938, respectively; 6,110,490, respectively; 6,534,484 and 7,687,070, the disclosures of each of which are incorporated herein by reference. In one embodiment, a method of transiently altering the expression of a protein in a TIL population comprises the use of U.S. patent No. 5,766,902; 6,025,337, respectively; 6,410,517, respectively; 6,475,994 and 7,189,705, the disclosures of each of which are incorporated herein by reference.

In some embodiments, the transient alteration in protein expression results in an increase in T memory stem cells (TSCMs). TSCM is an early progenitor of central memory T cells that have undergone antigen. TSCMs typically exhibit long-term survival, self-renewal and pluripotency that define stem cells, and are often required for the production of potent TIL products. TSCM has shown enhanced anti-tumor activity compared to other T cell subsets in a mouse model of adoptive cell transfer (Gattinoni et al, Nat Med 2009, 2011; Gattinoni, Nature rev. cancer, 2012; ciri et al, Blood 2013). In some embodiments, the transient change in protein expression causes the TIL population to have a composition comprising a high proportion of TSCMs. In some embodiments, the transient alteration in protein expression increases the percentage of TSCM by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the transient alteration in protein expression increases TSCM in the TIL population by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold. In some embodiments, the transient alteration in protein expression is such that the population of TILs has a TSCM of at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, the transient alteration in protein expression is such that the TSCM for the therapeutic TIL population is at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

In some embodiments, the transient alteration in protein expression causes T cells that are undergoing the antigen to become rejuvenated (rejuvenation). In some embodiments, restoring viability comprises, for example, increased proliferation, increased T cell activation, and/or increased antigen recognition.

In some embodiments, the transient alteration in protein expression alters expression in a majority of T cells to preserve a tumor-derived TCR repertoire (TCR retetoreire). In some embodiments, the transient alteration in protein expression does not alter the TCR repertoire of tumor origin. In some embodiments, the transient change in protein expression maintains a tumor-derived TCR repertoire.

In some embodiments, the transient alteration of the protein results in altered expression of a particular gene. In some embodiments, genes targeted by transient changes in protein expression include, but are not limited to, PD-1 (also referred to as PDCD1 or CC279), TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric costimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF β, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP protein kinase a (pka). In some embodiments, the gene targeted by the transient alteration of protein expression is selected from PD-1, TGFBR2, CCR4/5, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF β, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, and/or cAMP 39a (pka). In some embodiments, the transient alteration in protein expression targets PD-1. In some embodiments, the transient change in protein expression targets TGFBR 2. In some embodiments, the transient alteration of protein expression targets CCR 4/5. In some embodiments, the transient alteration in protein expression targets CBLB. In some embodiments, the transient alteration in protein expression targets CISH. In some embodiments, the transient alteration of protein expression targets CCR (chimeric co-stimulatory receptor). In some embodiments, the transient alteration in protein expression targets IL-2. In some embodiments, the protein expression transient change targeting IL-12. In some embodiments, the transient alteration in protein expression targets IL-15. In some embodiments, the transient alteration in protein expression targets IL-21. In some embodiments, the transient alteration in protein expression targets NOTCH 1/2 ICD.

In some embodiments, transient changes in protein expression target TIM 3. In some embodiments, the transient alteration in protein expression targets LAG 3. In some embodiments, the transient alteration in protein expression targets TIGIT. In some embodiments, the transient alteration in protein expression targets TGF β. In some embodiments, the transient alteration of protein expression targets CCR 1. In some embodiments, the transient alteration of protein expression targets CCR 2. In some embodiments, the transient alteration of protein expression targets CCR 4. In some embodiments, the transient alteration of protein expression targets CCR 5. In some embodiments, the transient alteration in protein expression targets CXCR 1. In some embodiments, the transient alteration in protein expression targets CXCR 2. In some embodiments, the transient alteration in protein expression targets CSCR 3. In some embodiments, the transient alteration in protein expression targets CCL2 (MCP-1). In some embodiments, the transient change in protein expression targets CCL3(MIP-1 α). In some embodiments, the transient change in protein expression targets CCL4(MIP1- β). In some embodiments, the transient alteration in protein expression targets CCL5 (RANTES). In some embodiments, the transient alteration in protein expression targets CXCL 1. In some embodiments, the transient alteration in protein expression targets CXCL 8. In some embodiments, the transient alteration in protein expression targets CCL 22. In some embodiments, the transient alteration in protein expression targets CCL 17. In some embodiments, the transient alteration in protein expression targets VHL. In some embodiments, the transient alteration in protein expression targets CD 44. In some embodiments, the transient alteration in protein expression targets PIK3 CD. In some embodiments, the transient alteration in protein expression targets SOCS 1. In some embodiments, the transient change in protein expression targets cAMP protein kinase a (pka).

In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of chemokine receptors. In some embodiments, chemokine receptors overexpressed by transient protein expression include receptors with ligands including, but not limited to, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1, CXCL8, CCL22, and/or CCL 17. In some embodiments, chemokine receptors overexpressed by transient protein expression include receptors with ligands including, but not limited to, IL-2, IL-7, IL-10, IL-15, and IL-21, and NOTCH1/2 intracellular domain (ICD). In some embodiments, the transient alteration in protein expression targets the NOTCH signaling pathway, e.g., by NOTCH1/2 ICD and/or by other NOTCH ligands, such as mDLL1 (see, e.g., Kondo, T. et al, NOTCH-mediated conversion of activated T cells memory-like Tcells for adaptive immunological therapy, Nature Communications, volume 8, article No.: 15338(2017), the entire contents of which are incorporated herein by reference).

In some embodiments, transient alterations in protein expression result in reduced and/or decreased expression of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, TGF β R2, and/or TGF β (including, for example, resulting in blockade of the TGF β pathway). In some embodiments, the transient alteration in protein expression results in reduced and/or decreased expression of CBLB (CBL-B). In some embodiments, the transient alteration in protein expression results in a decrease and/or reduction in CISH expression.

In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of a chemokine receptor, thereby, for example, improving TIL trafficking or movement to a tumor site. In some embodiments, the transient alteration in protein expression results in increased and/or overexpression of CCR (chimeric co-stimulatory receptor). In some embodiments, the transient alteration in protein expression results in increased and/or overexpression of a chemokine receptor selected from CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, and/or CSCR 3.

In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of an interleukin. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of an interleukin selected from IL-2, IL-12, IL-15, and/or IL-21.

In some embodiments, the transient alteration in protein expression targets the NOTCH signaling pathway, e.g., by NOTCH1/2 ICD and/or by other NOTCH ligands, such as mDLL1 (see, e.g., Kondo, T. et al, NOTCH-media conversion of activated T cells into stem cell memory-like T cells for adaptive immunization, Nature Communications, volume 8, article No.: 15338(2017), the entire contents of which are incorporated herein by reference). In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of NOTCH1/2 ICD. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of a NOTCH ligand (e.g., mlll 1). In some embodiments, the transient alteration in protein expression results in increased and/or over-expression of VHL. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of CD 44. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of PIK3 CD. In some embodiments, the transient alteration in protein expression results in an increase and/or overexpression of SOCS 1.

In some embodiments, the transient change in protein expression results in a decrease and/or a decrease in cAMP protein kinase a (pka) expression.

In some embodiments, the transient alteration in protein expression reduces and/or decreases expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression reduces and/or decreases the expression of two molecules selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression reduces and/or decreases expression of PD-1 and a molecule selected from LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the transient alteration in protein expression reduces and/or decreases expression of PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration in protein expression reduces and/or decreases the expression of PD-1 and one of LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the transient alteration in protein expression results in a decrease and/or reduction in expression of PD-1 and LAG 3. In some embodiments, the transient alteration in protein expression results in a decrease and/or reduction in the expression of PD-1 and CISH. In some embodiments, the transient alteration in protein expression results in reduced and/or decreased expression of PD-1 and CBLB. In some embodiments, the transient alteration in protein expression results in a reduction and/or decrease in expression of LAG3 and CISH. In some embodiments, the transient alteration in protein expression results in a reduction and/or decrease in expression of LAG3 and CBLB. In some embodiments, the transient alteration in protein expression results in a decrease and/or reduction in expression of CISH and CBLB. In some embodiments, the transient alteration in protein expression results in a decrease and/or reduction in the expression of TIM3 and PD-1. In some embodiments, the transient alteration in protein expression results in reduced and/or decreased expression of TIM3 and LAG 3. In some embodiments, the transient alteration in protein expression results in a reduction and/or decrease in the expression of TIM3 and CISH. In some embodiments, the transient alteration in protein expression results in a decrease and/or reduction in the expression of TIM3 and CBLB.

In some embodiments, an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted into the first TIL population, the second TIL population, or the harvested TIL population (e.g., increased adhesion molecule expression) by a gamma-retrovirus or lentivirus approach.

In some embodiments, the transient alteration in protein expression reduces and/or enhances expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof, and increases and/or enhances expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof. In some embodiments, the transient alteration in protein expression reduces and/or decreases expression of a molecule selected from the group consisting of PD-1, LAG3, TIM3, CISH, CBLB, and combinations thereof, and increases and/or enhances expression of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof.

In some embodiments, expression is reduced by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

In some embodiments, expression is increased by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 85%, about 90%, or about 95%. In some embodiments, expression is increased by at least about 80%. In some embodiments, expression is increased by at least about 85%. In some embodiments, expression is increased by at least about 90%. In some embodiments, expression is increased by at least about 95%. In some embodiments, expression is increased by at least about 99%.

In some embodiments, transient changes in protein expression are induced by treating the TIL with Transcription Factors (TFs) and/or other molecules capable of transiently changing protein expression in the TIL. In some embodiments, a microfluidic platform without SQZ vectors is utilized to deliver Transcription Factors (TFs) and/or other molecules capable of transiently altering protein expression intracellularly. Such methods demonstrating the ability to deliver proteins, including transcription factors, to a variety of primary human cells, including T cells, have been described (Sharei et al, PNAS 2013, and Sharei et al, PLOS ONE 2015 and Greisbeck et al, j.immunology2015, volume 195), including rapid methods of using microfluidic constriction (microfluidics condensation) to deform cells such that TF or other molecules enter the cells; see, e.g., international patent application publication nos. WO2013/059343a1, WO2017/008063a1, or WO2017/123663a1, or U.S. patent application publication nos. US2014/0287509a1, US2018/0201889a1, or US2018/0245089a1, all of which are incorporated herein by reference in their entirety. Methods as described in international patent application publication nos. WO2013/059343a1, WO2017/008063a1, or WO2017/123663a1, or U.S. patent application publication nos. US2014/0287509a1, US2018/0201889a1, or US2018/0245089a1 may be used with the present invention to expose a TIL population to Transcription Factors (TFs) and/or other molecules capable of inducing transient protein expression, wherein the TF and/or other molecules capable of inducing transient protein expression provide an increase in tumor antigen expression and/or an increase in the number of tumor antigen-specific T cells in the TIL population, resulting in reprogramming of the TIL population and an increase in the therapeutic efficacy of the reprogrammed TIL population as compared to an un-reprogrammed TIL population. In some embodiments, reprogramming results in an increase in a subpopulation of effector T cells and/or central memory T cells relative to an initial TIL population or a prior (i.e., prior to reprogramming) TIL population, as described herein.

In some embodiments, Transcription Factors (TF) include, but are not limited to TCF-1, NOTCH 1/2ICD, and/or MYB. In some embodiments, the Transcription Factor (TF) is TCF-1. In some embodiments, the Transcription Factor (TF) is NOTCH 1/2 ICD. In some embodiments, the Transcription Factor (TF) is MYB. In some embodiments, a Transcription Factor (TF) is administered with an induced pluripotent stem cell culture (iPSC), such as a commercially available KNOCKOUT serum replacement (Gibco/ThermoFisher), to induce additional TIL reprogramming. In some embodiments, a Transcription Factor (TF) is administered with the iPSC mixture to induce additional TIL reprogramming. In some embodiments, the Transcription Factor (TF) is administered without the iPSC mixture. In some embodiments, reprogramming results in an increase in the percentage of TSCM. In some embodiments, reprogramming results in an increase in the percentage of TSCM by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of TSCM.

In some embodiments, a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population comprises:

(i) Obtaining a first TIL population from a resected tumor of a patient;

(ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population;

In one embodiment, a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) Adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) performing a sterile electroporation step on the second TIL population; wherein the step of sterile electroporation mediates transfer of at least one short interfering RNA or one messenger RNA;

(f) standing the second TIL population for about 1 day;

(g) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (f) to step (g) occurs without opening the system;

(h) Harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL population is a therapeutic TIL population;

(i) transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

According to one embodiment, a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(d) Adding OKT-3 to produce a second TIL population; wherein the first amplification is carried out in a closed vessel providing a first gas permeable surface region; wherein the first amplification is performed for about 1 to 3 days to obtain a second TIL population; wherein the number of second TIL groups is at least 50 times greater than the number of first TIL groups; wherein the transition from step (c) to step (d) occurs without opening the system;

(e) performing an SQZ microfluidic membrane disruption step on the second TIL population; wherein the SQZ microfluidic membrane disruption step mediates transfer of at least one short interfering RNA or one messenger RNA;

(f) standing the second TIL population for about 1 day;

(g) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (f) to step (g) occurs without opening the system;

(h) harvesting the therapeutic TIL population obtained from step (g) to provide a harvested TIL population; wherein the transition from step (g) to step (h) occurs without opening the system; wherein the harvested TIL population is a therapeutic TIL population;

(i) Transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

wherein the SQZ microfluidic membrane disruption step comprises delivering a short interfering RNA to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3) and combinations thereof.

In some embodiments, the methods of transiently altering protein expression as described above may be combined with methods of genetically modifying TIL populations, including the step of stably incorporating genes for producing more than one protein. In one embodiment, the method of genetically modifying a TIL population comprises the step of retroviral transduction. In one embodiment, the method of genetically modifying a TIL population comprises the step of lentiviral transduction. Lentiviral transductant lines are known in the art and are described, for example, in Levine et al, Proc.nat' l Acad.Sci.2006,103, 17372-77; zufferey et al, nat. Biotechnol.1997,15,871-75; dull et al, j.virology 1998,72,8463-71 and U.S. patent No. 6,627,442, the respective disclosures of which are incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population comprises the step of gamma-retroviral transduction. Gamma-retroviral transduction lines are known in the art and are described, for example, in Cepko and Pear, cur. prot. mol. biol.1996,9.9.1-9.9.16, the disclosure of which is incorporated herein by reference. In one embodiment, the method of genetically modifying a TIL population includes the step of transposon-mediated gene transfer. Transposon-mediated gene transfer systems are known in the art and include the following: wherein the transposase is provided in the form of a DNA expression vector or an expressible RNA or protein such that the transposase is not expressed in the transgenic cell for a long period of time, such as a transposase provided in the form of an mRNA (e.g., an mRNA comprising a cap and a poly-a tail). Suitable transposon-mediated gene transfer systems include salmon-type Tel-like transposases (SB or Sleeping Beauty transposases), such as SB10, SB11, and SB100x, and engineered enzymes with increased enzymatic activity, as described, for example, in Hackett et al, mol.

In one embodiment, a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) performing a sterile electroporation step or an SQZ microfluidic membrane disruption step on the second TIL population; wherein the sterile electroporation step or the SQZ microfluidic membrane disruption step mediates transfer of at least one short interfering RNA or one messenger RNA;

(f) standing the second TIL population for about 1 day;

(g) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (f) to step (g) occurs without opening the system;

(i) transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

wherein the electroporating step comprises delivering a short interfering RNA to inhibit expression of a molecule selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof; further wherein an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted into the first TIL population, the second TIL population, or the harvested TIL population by a gamma-retroviral approach or a lentiviral approach.

In some embodiments, the transient alteration in protein expression is a decrease in expression induced by self-delivering RNA interference (sd-RNA), which has a high percentage of 2' -OH substitutions (typically fluorine or-OCH) ₃) The chemically synthesized asymmetric siRNA duplex of (a), said sd-RNA comprising a sense (guide) strand of 20 nucleotides and a sense (satellite) strand of 13 to 15 bases conjugated to cholesterol at its 3' end using a Tetraethyl Ethylene Glycol (TEG) linker. Methods of using sd-RNA have been described in Khvorova and Watts, nat. Biotechnol.2017,35, 238-248; byrne et al, J.Ocul.Pharmacol.Ther.2013,29, 855-864; and Ligtenberg et al, mol. In one embodiment, delivery of sd-RNA to the TIL population is accomplished without electroporation, SQZ, or other methods, but using the following steps: the TIL population was exposed to sd-RNA at a concentration of 1. mu.M/10,000 TIL in the medium for 1 to 3 days. In certain embodiments, delivery of sd-RNA to a TIL population is accomplished using the following steps: the TIL population was exposed to sd-RNA at a concentration of 10. mu.M/10,000 TIL in the medium for 1 to 3 days. In one embodiment, delivery of sd-RNA to the TIL population is accomplished using the following steps: the TIL population was exposed to sd-RNA at a concentration of 50. mu.M/10,000 TIL in the medium for 1 to 3 days. In one embodiment, delivery of sd-RNA to the TIL population is accomplished using the following steps: the TIL population was exposed to sd-RNA at concentrations ranging from 0.1. mu.M/10,000 TIL to 50. mu.M/10,000 TIL in culture medium for 1 to 3 days. In one embodiment, delivery of sd-RNA to the TIL population is accomplished using the following steps: exposing the TIL population to sd-RNA at a concentration of 0.1 μ Μ/10,000TIL to 50 μ Μ/10,000TIL in the culture medium for 1 to 3 days; wherein the exposure to sd-RNA is performed two, three, four or five times by adding fresh sd-RNA to the medium. Other suitable methods are described in For example, U.S. patent application publication numbers US2011/0039914a1, US2013/0131141a1, and US2013/0131142a1, and U.S. patent number 9,080,171, the disclosures of which are incorporated herein by reference.

In some embodiments, sd-RNA is inserted into the TIL population during production using the method of fig. 32. In some embodiments, sd-RNA encodes RNA that interferes with NOTCH 1/2 ICD, NOTCH ligand mDLLl, PD-1, CTLA-4TIM-3, LAG-3, TIGIT, TGF β, TGFBR2, cAMP Protein Kinase A (PKA), BAFF BR3, CISH, and/or CBLB. In some embodiments, the reduction in expression is determined based on the percentage of gene silencing, for example as assessed by flow cytometry and/or qPCR. In some embodiments, expression is reduced by about 5%, about 10%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%, about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 85%, about 90%, or about 95%. In some embodiments, expression is reduced by at least about 80%. In some embodiments, expression is reduced by at least about 85%. In some embodiments, expression is reduced by at least about 90%. In some embodiments, expression is reduced by at least about 95%. In some embodiments, expression is reduced by at least about 99%.

In one embodiment, a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, one or more self-delivering RNAs (sd-RNA), optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (f) to step (g) occurs without opening the system;

(i) transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

In one embodiment, a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population comprises:

(a) obtaining a first TIL population from a tumor resected from a patient by processing a tumor sample obtained from the patient into a plurality of tumor fragments;

(b) adding tumor fragments to the closed system;

(c) performing a first expansion by culturing the first TIL population in cell culture medium comprising IL-2 and optionally a 4-1BB agonist antibody for about 2 to 5 days;

(e) Generating a third TIL population by a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, one or more self-delivering RNAs (sd-RNA), optionally an OKT-3 antibody, optionally an OX40 antibody, and Antigen Presenting Cells (APCs); wherein the second amplification is performed for about 7 days to 11 days to obtain a third population of TILs; wherein the second amplification is carried out in a closed vessel providing a second gas permeable surface region; wherein the transition from step (f) to step (g) occurs without opening the system;

(i) transferring the TIL population harvested from step (e) to an infusion bag; wherein the transition from step (e) to step (f) occurs without opening the system; and

(j) cryopreserving the harvested TIL population using a dimethylsulfoxide-based cryopreservation medium;

wherein the expression of one or more sd-RNA transient inhibitory molecules selected from the group consisting of PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3) and combinations thereof; wherein an adhesion molecule selected from the group consisting of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, and combinations thereof is inserted into the first TIL population, the second TIL population, or the harvested TIL population by a gamma-retroviral approach or a lentiviral approach.

sd-RNA method

Self-delivery RNAi techniques based on chemical modification of siRNA can be used with the methods of the invention to successfully deliver sd-RNA to the TILs described herein. The binding of backbone modified asymmetric siRNA structures to hydrophobic ligands (see, e.g., Ligtenberg et al, mol. therapy, 2018 and US20160304873, and figures 36 and 37 herein) allows sd-RNA to penetrate into cultured mammalian cells using nuclease stability of sd-RNA by simple addition to culture medium without the need for other formulations and methods. Such stability can support a constant level of RNAi-mediated reduction in target gene activity by merely maintaining the active concentration of sd-RNA in the culture medium. Without being bound by theory, backbone stabilization of sd-RNA can prolong the reduction of gene expression effects, which can last for months in non-dividing cells.

In one embodiment, the sd-RNA used herein that targets a gene disclosed herein has the structure shown in figure 36 or figure 37.

In some embodiments, greater than 95% of TIL transfection efficiency and reduced target expression of various specific sd-RNAs occurs. In some embodiments, sd-RNA containing several unmodified ribose residues is replaced with a fully modified sequence to increase the titer and/or longevity of the RNAi effect. In some embodiments, the reduction in expression effect is maintained for 12 hours, 24 hours, 36 hours, 48 hours, 5 days, 6 days, 7 days, or 8 days or more. In some embodiments, the effect of decreased expression is decreased more than 10 days after sd-RNA treatment of TIL. In some embodiments, the reduction in expression to maintain expression of interest is greater than 70%. In some embodiments, the reduction in expression to maintain target expression of TIL is greater than 70%. In some embodiments, the reduction in expression in the PD-1/PD-L1 pathway allows TIL to exhibit a more potent in vivo effect, in some embodiments due to the avoidance of inhibition of the PD-1/PD-L1 pathway. In some embodiments, a decrease in PD-1 expression by sd-RNA results in an increase in TIL proliferation.

Selection and characterization of sd-RNA

sd-RNA oligonucleotide structures

Small interfering RNAs (siRNA), sometimes also referred to as short interfering RNAs or silencing RNAs, are double-stranded RNA molecules, typically 19 to 25 base pairs in length. sirnas are used for RNA interference (RNAi), in which the siRNA interferes with the expression of a specific gene having a complementary nucleotide sequence.

Double-stranded dna (dsrna) can generally be used to define any molecule comprising a pair of complementary strands of RNA, which are typically the sense (follower) and antisense (leader) strands, and can include a single-stranded overhang region. In contrast to siRNA, the term dsRNA generally refers to a precursor molecule comprising a sequence of an siRNA molecule that is released from a larger dsRNA molecule by the action of a cleavage enzyme system (including Dicer).

sd-RNA (self-delivering RNA) is a new class of covalently modified RNAi compounds that do not require a delivery vehicle for entry into cells and have improved pharmacology compared to traditional siRNA. "self-delivering RNA" or "sd-RNA" is a hydrophobically modified RNA interference-antisense hybrid that has proven to be highly effective in vitro in primary cells and in vivo when administered topically. Non-toxic robust uptake and/or silencing has been demonstrated. sd-RNA is typically an asymmetrically chemically modified nucleic acid molecule with a minimal double-stranded region. sd-RNA molecules typically comprise a single-stranded region and a double-stranded region, and may comprise a variety of chemical modifications within the single-stranded and double-stranded regions of the molecule. Furthermore, as described herein, sd-RNA molecules can be linked to hydrophobic conjugates, e.g., molecules of the conventional and higher sterol type. sd-RNA (and/or RNA that can be used in a manner similar to sd-RNA) and related methods of making such sd-RNA have also been described extensively in, for example, U.S. patent publication No. US2016/0304873, international patent application publication No. WO2010/033246, international patent application publication No. WO2017/070151, international patent application publication No. WO2009/102427, international patent application publication No. WO201/1119887, international patent application publication No. WO2010/033247, international patent application publication No. WO2009045457, international patent application publication No. WO2011/119852, international patent application publication No. WO2011/119871, U.S. patent publication No. US2011/0263680, international patent application publication No. WO2010/033248, international patent application publication No. WO2010/078536, international patent application publication No. WO2010/090762, U.S. US patent publication No. US20110039914, international patent publication No. WO2011/109698, international patent application publication No. WO2010/090762, U.S. patent No. US8,815,818, international patent application publication No. WO2016/094845, international patent application publication No. WO2017/193053, U.S. patent publication No. US2006/0276635, international patent application publication No. WO2001/009312, U.S. patent publication No. US2017/0043024, U.S. patent publication No. US2017/0312367, U.S. patent publication No. US2016/0319278, U.S. patent publication No. US2017/0369882, U.S. patent No. US8,501,706, U.S. patent No. US2004/0224405, U.S. patent No. US8,252,755, U.S. patent No. US2007/0031844, U.S. patent No. US2007/0039072, U.S. patent publication No. US2007/0207974, U.S. patent publication No. US2007/0213520, U.S. patent publication No. US/2007/0213521, U.S. patent publication No. US2007/0219362, U.S, U.S. patent publication No. US2014/0148362, U.S. patent publication No. US2016/0193242, U.S. patent publication No. US2016/01946461, U.S. patent publication No. US2016/0201058, U.S. patent publication No. US2016/0201065, U.S. patent publication No. US2017/0349904, U.S. patent publication No. US2018/0119144, U.S. patent No. US7,834,170, U.S. patent No. US8,090,542, and U.S. patent publication No. US2012/0052487, the entire contents of which are incorporated herein by reference for all purposes; sd-RNA is also commercially available from Advirna LLC (Worcester, MA, USA). To optimize the structure, chemistry, targeting location, sequence preference, etc., of sd-RNA, proprietary algorithms have been developed and used for sd-RNA titer prediction (see e.g., US 20160304873). Based on these analyses, functional sd-RNA sequences are generally defined as a reduction in expression of more than 70% at a concentration of 1 μ M with a probability of more than 40%.

sd-RNA oligonucleotide structures

In some embodiments, more than one sd-RNA for use in the invention can be generated from a linear double stranded DNA template. In some embodiments, the linear double stranded DNA template used to generate more than one sd-RNA is a template as described in U.S. patent No. US8,859,229 and described below.

In some embodiments, a linear double stranded DNA template obtained by Polymerase Chain Reaction (PCR) and suitable for in vitro transcription of mRNA comprises, from 5 'to 3': an RNA polymerase promoter on the coding strand of double-stranded DNA, a 5' untranslated region (less than 3,000 nucleotides in length effective to translate mRNA into a detectable polypeptide upon transfection into a eukaryotic cell), an open reading frame encoding the polypeptide; wherein the polypeptide is heterologous to the cell to be transfected; wherein the polypeptide is selected from: ligands or receptors for immune cells, polypeptides that stimulate or inhibit immune system function, polypeptides that inhibit the function of oncogenic polypeptides, a 3' untranslated region that can efficiently translate mRNA into a detectable polypeptide upon transfection into eukaryotic cells, and a poly (a) stretch of 50 to 5,000 nucleotides on the coding strand of double-stranded DNA; wherein the promoter is heterologous to the open reading frame; wherein the DNA template is not contained in the DNA vector and terminates at the 3' end of the poly (A) stretch. In some embodiments, the RNA polymerase promoter comprises a consensus binding sequence for an RNA polymerase selected from T7, T3, or SP6 RNA polymerase. In some embodiments, the open reading frame encodes a fusion polypeptide. In some embodiments, the open reading frame encodes a polypeptide selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2 ICD, TIM3, LAG3, TIGIT, TGF beta, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 alpha), CCL4(MIP 1-beta), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP Protein Kinase A (PKA), and combinations thereof. In some embodiments, the open reading frame encodes a polypeptide selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3) and combinations thereof. In some embodiments, the linear double stranded DNA template further comprises an internal ribosome entry site. In some embodiments, the length of the poly (A) stretch is 300-400 nucleotides.

In some embodiments, the linear double stranded DNA template of claim 1; wherein the template consists of, from 5 'to 3': an RNA polymerase promoter on the coding strand of double-stranded DNA, a 5' untranslated region (less than 3,000 nucleotides in length effective to translate mRNA into a detectable polypeptide upon transfection into a eukaryotic cell), an open reading frame encoding the polypeptide; wherein the polypeptide is heterologous to the cell to be transfected; wherein the polypeptide is selected from: ligands or receptors for immune cells, polypeptides that stimulate or inhibit immune system function, polypeptides that inhibit the function of oncogenic polypeptides, a 3' untranslated region that can efficiently translate mRNA into a detectable polypeptide upon transfection into eukaryotic cells, and a poly (a) stretch of 50 to 5,000 nucleotides on the coding strand of double-stranded DNA; wherein the promoter is heterologous to the open reading frame; wherein the DNA template is not contained in the DNA vector and terminates at the 3' end of the poly (A) stretch. In some embodiments, the 3' untranslated region is at least 100 nucleotides in length.

In some embodiments, the present invention provides a method of producing the linear double-stranded DNA template described above; wherein, the method comprises the following steps: generating a forward primer and a reverse primer; wherein the forward primer comprises: a plurality of nucleotides substantially complementary to the non-coding strand of the target double-stranded DNA of interest, and a plurality of nucleotides that serve as binding sites for RNA polymerase; wherein the reverse primer comprises: a plurality of nucleotides substantially complementary to the coding strand of the target double-stranded DNA of interest, and a plurality of deoxythymidine nucleotides; and performing polymerase chain reaction amplification on the target DNA by using the forward primer and the reverse primer to form a linear double-stranded DNA template. In some embodiments, the present invention provides a method of producing the linear double stranded DNA template described above, wherein the method comprises: generating a forward primer and a reverse primer; wherein the forward primer comprises a plurality of nucleotides that are substantially complementary to a region of nucleotides directly upstream of the target double-stranded DNA of interest; wherein the reverse primer comprises a plurality of nucleotides that are substantially complementary to a nucleotide region immediately downstream of the target double-stranded DNA of interest; and performing polymerase chain reaction amplification on the target DNA by using the forward primer and the reverse primer to form a linear double-stranded DNA template. In some embodiments, the primer comprises a nucleotide sequence that is substantially complementary to stretches of nucleotides (stretches of nucleotides) in the 5 'untranslated region and the 3' untranslated region of the double-stranded DNA of interest. In some embodiments, the primer comprises a nucleotide sequence that is substantially complementary to a stretch of nucleotides within an open reading frame of the double-stranded DNA of interest. In some embodiments, the primer comprises a nucleotide sequence that is substantially complementary to a stretch of nucleotides within an open reading frame of the double-stranded DNA of interest; wherein the primer further comprises a nucleotide segment comprising a 5 'untranslated region and a 3' untranslated region; wherein the stretch of nucleotides in the forward primer comprising the 5' untranslated region is between the nucleotides comprising the RNA polymerase promoter and the nucleotides substantially complementary to the non-coding strand of the target double stranded DNA of interest; wherein the stretch of nucleotides in the reverse primer comprising the 3' untranslated region is between the plurality of deoxythymidine nucleotides and the nucleotides substantially complementary to the coding strand of the target double-stranded DNA of interest. In some embodiments, the forward primer and open reading frame comprise a consensus Kozak sequence.

In some embodiments, the invention provides methods of producing more than one RNA for transfecting a cell, the methods comprising in vitro transcription from a linear double stranded DNA template. In some embodiments, the method further comprises extending the poly (a) tail of the RNA with one or more adenine nucleotides or analogs thereof using poly (a) polymerase. In some embodiments, the method further comprises adding a nucleotide during transcription that serves as a 5' cap for the RNA that is transcribed. In some embodiments, the RNA targets a polypeptide selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCR (chimeric co-stimulatory receptor), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM3, LAG3, TIGIT, TGF beta, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 alpha), CCL4(MIP 1-beta), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP Protein Kinase A (PKA), and combinations thereof. In some embodiments, the RNA targets a polypeptide selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3) and combinations thereof.

In some embodiments, the invention uses the use of one or more isolated RNAs produced from a linear double-stranded DNA template and comprising one or more open reading frames. In some embodiments, the invention provides a method of expressing one or more RNAs in a cell, the method comprising contacting the cell with one or more RNAs produced from a linear double-stranded DNA template. In some embodiments, the RNA is present in unequal molar amounts in the cell to provide different expression levels of the RNA. In some embodiments, the one or more RNAs target a polypeptide selected from the group consisting of: PD-1, TGFBR2, CBLB (CBL-B), CISH, CCRs (chimeric costimulatory receptors), IL-2, IL-12, IL-15, IL-21, NOTCH 1/2ICD, TIM3, LAG3, TIGIT, TGF beta, CCR2, CCR4, CCR5, CXCR1, CXCR2, CSCR3, CCL2(MCP-1), CCL3(MIP-1 alpha), CCL4(MIP 1-beta), CCL5(RANTES), CXCL1/CXCL8, CCL22, CCL17, CXCL1/CXCL8, VHL, CD44, PIK3CD, SOCS1, cAMP Protein Kinase A (PKA), and combinations thereof. In some embodiments, the one or more RNAs target a polypeptide selected from the group consisting of: PD-1, LAG-3, TIM-3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3) and combinations thereof.

(i) Untranslated regions

Chemical structures that have the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has a 5'UTR and a 3' UTR. The examples below show that inclusion of a 44 base pair 5'UTR in a PCR template results in more efficient translation of transcribed CFP RNA than a PCR template containing only a 6 base pair 5' UTR. The examples also show that the addition of a 113 base pair 3'UTR results in a higher translation efficiency of the transcribed GFP RNA compared to a PCR template comprising only an 11 base pair 3' UTR. Typically, the 3'UTR is more than 100 nucleotides in length, so 3' UTRs longer than 100 nucleotides are preferred. In one embodiment, the 3' UTR sequence is from 100 to 5000 nucleotides. The length of the 5' UTR is less important than the length of the 3' UTR, and the length of the 5' UTR can be shorter. In one embodiment, the 5' UTR is 0 to 3000 nucleotides in length. The length of the 5'UTR and 3' UTR sequences to be added to the coding region can be varied by different methods including, but not limited to, designing PCR primers for annealing to different UTR regions. Using this method, one of ordinary skill in the art can modify the desired 5'UTR and 3' UTR lengths after transfection of the transcribed RNA to achieve optimal translation efficiency.

The 5'UTR and 3' UTR may be naturally occurring endogenous 5 'UTRs and 3' UTRs of the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by adding UTR sequences to the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be used to alter RNA stability and/or translation efficiency. For example, AU-rich elements in the 3' UTR sequence are known to reduce mRNA stability. Thus, based on the well-known properties of UTRs in the art, the 3' UTRs can be selected or designed to increase the stability of transcribed RNA.

In one embodiment, the 5' UTR may comprise a Kozak sequence of an endogenous gene. Alternatively, when a non-endogenous 5'UTR of the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5' UTR sequence. Kozak sequences may improve the translation efficiency of some RNA transcripts, but it does not appear that all RNAs require Kozak sequences for efficient translation. The requirement for Kozak sequence for many mrnas is known in the art. In other embodiments, the 5' UTR may be from an RNA virus whose RNA genome is stable in the cell. In other embodiments, various nucleotide analogs can be used in the 3'UTR or 5' UTR to prevent exonuclease degradation of the mRNA.

(ii) RNA polymerase promoter

In order to synthesize RNA from a DNA template without gene cloning, a transcription promoter should be ligated to the DNA template upstream of the sequence to be transcribed. The bacteriophage RNA polymerase promoter sequence may be linked to the St UTR by different genetic engineering methods (e.g. DNA ligation), or the bacteriophage RNA polymerase promoter sequence may be added to the forward primer (5') of a sequence substantially complementary to the target DNA. When a sequence acting as an RNA polymerase promoter is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is the T7 polymerase promoter as described above. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

(iii) Poly (A) tail and 5' cap

In a preferred embodiment, the mRNA has caps at both the 5 'end and the 3' poly (a) tail that determine ribosome binding, translation initiation, and mRNA stability in the cell. On circular DNA templates (e.g., plasmid DNA), RNA polymerase produces long concatamer (concatameric) products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR produces mRNA of normal size, which does not function in eukaryotic transfection even if it is polyadenylated after transcription.

On a linear DNA template, phage T7RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc. acids Res.,13:6223-36 (1985); Nacheva and Biochem. et al., 270:1485-65 (2003)). This may lead to bending of the runaway transcript (runofftranscript) which then exchanges with the template of the second DNA strand or transcription of the RNA itself (Triana-Alonso et al, J.biol. chem.,270: 6298-; 307 (1995); Dunn and Studier, J.mol. biol.,166: 477-; 535 (1983); Arnaud-Barbe et al, Nuc.acids Res.,26:3550-54 (1998); Macdonald et al, 1993)), which then leads to reverse aberrant transcription and accumulation of double stranded RNA, thereby inhibiting gene expression. DNA linearization is not sufficient by itself for correct transcription (Triana-Alonso et al, J.biol. chem.,270:6298-307 (1995); Dunn and Studier, J.mol. biol.,166:477-535 (1983); Arnaud-Barbe et al, 1998Nuc.acids Res.,26:3550-54 (1998); Macdonald et al, J.mol. biol.,232:1030-47 (1993); Nakano et al, Biotechn. Bioeng.,64:194-99(1999)), DNA linearized downstream of a 64 to 100 nucleotide poly (A/T) segment yields a good template (Saeboe-Larssen et al, J.Imnon. meth., 191: 203 (2002); czkoki et al, Cancer: 1028, 2000: 14; Riopgo et al, 1028: 14: 53, 2000). The endogenous termination signal of T7RNA polymerase encodes RNA that folds into a stem-loop structure following the uridine residue tracing (Dunn and J.mol.biol.,166:477-535 (1983); Arnaud-Barbe et al, 1998Nuc.acids Res.,26:3550-54 (1998)). Even without the hairpin, the synthetic uridine traces attenuated transcription (Kiyama and Oishi, Nuc. AcidsRees., 24:4577-4583 (1996)). It is hypothesized that plasmid DNA linearization downstream of the poly (A/T) stretch may form a "dynamic" terminator that prevents potentially aberrant transcription: 3' extension and reverse transcription of RNA transcripts in the poly (A/T) tract will generate an ever-increasing terminal-like signal-the extended poly (U) tract and the poly (A/U) hairpin. Thus, reverse PCR primers were designed with a 3 'anchor sequence downstream of the GFP gene and 5' of the 100 base stretch of poly (T) (FIG. 38).

The conventional method for integration of a polyA/T stretch into a DNA template is molecular cloning. However, the polyA/T sequences incorporated into plasmid DNA can lead to plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated by deletions and other aberrations. This makes the cloning process time consuming, laborious and often unreliable. This is why a method is highly desired which can construct a DNA template having a polyA/T3' stretch without cloning.

The polyA/T segment of the transcribed DNA template may be generated during PCR by using a reverse primer containing a polyT tail, such as a 100T tail (which may be 50T to 5000T in size), or by any other method after PCR, including but not limited to DNA ligation or in vitro recombination. The poly (A) tail may also provide stability to the RNA and reduce degradation of the RNA. Generally, the length of the poly (A) tail is positively correlated with the stability of the transcribed RNA. In one embodiment, the poly (a) tail is 100 to 5000 adenosines. The examples below show that a 100 base pair poly (A) stretch is sufficient for efficient translation of RNA transcripts.

After in vitro transcription, the poly (A) tail of the RNA may be further extended using a poly (A) polymerase, such as E.coli polyA polymerase (E-PAP). The examples below show that increasing the length of the poly (A) tail from 100 nucleotides to 300 to 400 nucleotides increases the translation efficiency of the RNA by about two-fold. Furthermore, attaching different chemical groups to the 3' end can increase the stability of the mRNA. Such linkages may include modified/artificial nucleotides, aptamers, and other compounds. For example, an ATP analog can be added to a poly (A) tail using a poly (A) polymerase. ATP analogues can further improve the stability of RNA. Suitable ATP analogues include, but are not limited to, cordiiocipin and 8-azaadenosine.

The 5' cap can also provide stability to the RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein comprises a 5' cap. For example, the 5' cap may be m⁷G(5')ppp(5')G、m⁷G (5') ppp (5') A, G (5') ppp (5') G or G (5') ppp (5') A ' cap analogs, all of which are commercially available. The 5' cap may also be an anti-reverse cap analog (ARCA, anti-reverse-cap-analog) (see Stepinski et al, RNA,7:1468-95(2001)) or any other suitable analog. The 5' cap is provided using techniques known in the art and described herein (Cougot et al, Trends in biochem. Sci.,29:436- & 444 (2001); Stepinski et al, RNA,7:1468-95 (2001); Elango et al, Biochim. Biophys. Res. Commun.,330:958- & 966 (2005)).

The RNA produced by the methods disclosed herein can further comprise an Internal Ribosome Entry Site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA and facilitates initiation of translation. May comprise any solute suitable for electroporation of cells, which may comprise factors that promote cell permeability and viability, such as carbohydrates, polypeptides, lipids, proteins, antioxidants, and surfactants.

In some embodiments, the sd-RNA sequence used in the present invention shows a 70% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in the invention shows a 75% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in the invention shows an 80% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in the present invention shows 85% reduction in target gene expression. In some embodiments, the sd-RNA sequences used in the invention show a 90% reduction in target gene expression. In some embodiments, the sd-RNA sequence used in the invention shows a 95% reduction in target gene expression. In some embodiments, the sd-RNA sequences used in the invention show a 99% reduction in target gene expression. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 0.25 μ Μ to about 10 μ Μ (in some embodiments, about 0.25 μ Μ to about 4 μ Μ). In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 0.25 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 0.5 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 0.75 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.25 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.5 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.75 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.25 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.5 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.75 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.25 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.5 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.75 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 4.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 5.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 6.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 7.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 8.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 9.0 μ Μ. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 10.0 μ Μ. In some embodiments, sd-RNA sequences used in the invention exhibit reduced expression of a target gene when delivered at a concentration of about 0.25 μ Μ/10,000TIL to about 10 μ Μ/10,000TIL or about 0.25 μ Μ/10,000TIL to about 4 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 0.25 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 0.5 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 0.75 μ M/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.25 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.5 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 1.75 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.25 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.5 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 2.75 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.25 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.5 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.75 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 4.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 5.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 6.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 7.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 8.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 9.0 μ Μ/10,000 TIL. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 10.0 μ Μ/10,000 TIL. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 0.25. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 0.5. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 0.75. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 1.0. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 1.25. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 1.5. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 1.75. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 2.0. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 2.25. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 2.5. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 2.75. mu.M/10,000 TIL/100. mu.L of medium. The sd-RNA sequences used in the present invention show reduced expression of the target gene when delivered at a concentration of about 3.0. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.25 μ Μ/10,000TIL/100 μ Μ medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.5. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 3.75 μ M/10,000TIL/100 μ L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 4.0. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 5.0. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 6.0. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 7.0. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 8.0. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 9.0. mu.M/10,000 TIL/100. mu.L of medium. In some embodiments, the sd-RNA sequences used in the invention show reduced expression of the target gene when delivered at a concentration of about 10.0. mu.M/10,000 TIL/100. mu.L of medium.

sd-RNA modification

In some embodiments, the oligonucleotide agent comprises one or more modifications to increase the stability and/or effectiveness of the therapeutic agent and to achieve effective delivery of the oligonucleotide to the cell or tissue to be treated. Such modifications may include 2' -O-methyl modifications, 2' -O-fluoro modifications, phosphorodithioate modifications, 2' F modified nucleotides, 2' -O-methyl modified nucleotides, and/or 2' deoxynucleotides. In some embodiments, the oligonucleotide is modified to include one or more hydrophobic modifications, including, for example, sterol, cholesterol, vitamin D, naphthyl, isobutyl, benzyl, indole, tryptophan, and/or phenyl. In another specific embodiment, the chemically modified nucleotide is a combination of phosphorothioate, 2 '-O-methyl, 2' deoxy, hydrophobic modification and phosphorothioate. In some embodiments, the saccharide can be modified, and the modified saccharide can include, but is not limited to, D-ribose, 2' -O-alkyl (including 2' -O-methyl and 2' -O-ethyl), i.e., 2' -alkoxy, 2' -amino, 2' -S-alkyl, 2' -halo (including 2' -fluoro), T-methoxyethoxy, 2' -allyloxy (-OCH), and the like₂CH＝CH₂) 2 '-propargyl, 2' -propyl, ethynyl, ethenyl, propenyl, cyano and the like. In one embodiment, the sugar moiety may be a hexose and is incorporated into the oligonucleotide as described (Augustyns, K. et al, Nucl. acids. Res.18: 4711 (1992)).

In some embodiments, the double-stranded oligonucleotides of the invention are double-stranded over their entire length, i.e., have no overhanging single-stranded sequence at either end of the molecule, i.e., are blunt-ended. In some embodiments, each nucleic acid molecule can have a different length. In other words, the double-stranded oligonucleotide of the present invention is not double-stranded over its entire length. For example, when two separate nucleic acid molecules are used, one of the molecules (e.g., a first molecule comprising an antisense sequence) may be longer (resulting in a portion of the molecule being single stranded) than a second molecule that hybridizes to it. In some embodiments, when a single nucleic acid molecule is used, a portion of the molecule at either end may remain single stranded.

In some embodiments, a double-stranded oligonucleotide of the invention contains mismatches and/or loops or bulges, but at least about 70% of the length of the oligonucleotide is double-stranded. In some embodiments, a double-stranded oligonucleotide of the invention is double-stranded over at least about 80% of the length of the oligonucleotide. In another embodiment, a double-stranded oligonucleotide of the invention is double-stranded over at least about 90% -95% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention is double-stranded over at least about 96-98% of the length of the oligonucleotide. In some embodiments, a double-stranded oligonucleotide of the invention comprises at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.

In some embodiments, oligonucleotides can be protected, for example, by modifying the 3 'or 5' linkage, from nucleases (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526). For example, oligonucleotides can be rendered resistant by the inclusion of "blocking groups". As used herein, the term "blocking group" as used herein refers to a substituent (e.g., a substituent other than an OH group) that may be attached to an oligonucleotide or a core monomer as a protecting or coupling group for synthesis (e.g., FITC, propyl (CH)₂-CH₂-CH₃) Ethylene glycol (-O-CH)₂-CH₂-O-), Phosphate (PO)₃ ²⁺) Phosphonate or phosphoramidite) "blocking groups" may also include "terminal blocking groups" or "exonuclease blocking groups" that protect the 5 'and 3' ends of oligonucleotides, including modified nucleotide and non-nucleotide exonuclease resistant structures.

In some embodiments, at least a portion of consecutive polynucleotides within the sd-RNA are linked by surrogate bonds (e.g., phosphorothioate bonds).

In some embodiments, the chemical modification can result in an increase in cellular uptake of at least 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500. In some embodiments, at least one of the C or U residues comprises a hydrophobic modification. In some embodiments, a plurality of C and U comprise hydrophobic modifications. In some embodiments, at least 10%, 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or at least 95% of C and U may comprise hydrophobic modifications. In some embodiments, all C and U comprise a hydrophobic modification.

In some embodiments, the sd-RNA or sd-rxRNA exhibits enhanced endosomal release of the sd-rxRNA molecule through incorporation of a protonatable amine. In some embodiments, a protonatable amine is incorporated into the sense strand (in a portion of the molecule that is discarded after RISC loading). In some embodiments, the sd-RNA compounds of the invention comprise an asymmetric compound comprising a duplex region (required for a 10-15 base long effective RISC entry) and a single stranded region of 4-12 nucleotides in length; a duplex having 13 nucleotides. In some embodiments, a 6 nucleotide single stranded region is employed. In some embodiments, the single-stranded region of the sd-RNA comprises 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). In some embodiments, 6-8 phosphorothioate internucleotide linkages are employed. In some embodiments, the sd-RNA compounds of the invention also include unique chemical modification patterns that provide stability and are compatible with RISC entries.

For example, the guide chain may also be modified by any chemical modification that consolidates stability without interfering with RISC entries. In some embodiments, the chemical modification pattern in the guide strand comprises: most of the C and U nucleotides are 2'F modified and the 5' end is phosphorylated.

In some embodiments, at least 30% of the nucleotides in the sd-RNA or sd-rxRNA are modified. In some embodiments, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nucleotides in the sd-RNA or sd-rxRNA are modified. In some embodiments, 100% of the nucleotides in the sd-RNA or sd-rxRNA are modified.

In some embodiments, the sd-RNA molecule has a minimal double-stranded region. In some embodiments, the double-stranded region of the molecule is 8-15 nucleotides in length. In some embodiments, the double-stranded region of the molecule is 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides in length. In some embodiments, the double-stranded region is 13 nucleotides long. There may be 100% complementarity between the guide strand and the follower strand, or there may be more than one mismatch between the guide strand and the follower strand. In some embodiments, at one end of a double-stranded molecule, the molecule is blunt-ended or has a one nucleotide overhang. In some embodiments, the single-stranded region of the molecule is 4-12 nucleotides long. In some embodiments, the single-stranded region can be 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides in length. In some embodiments, the single-stranded region may also be less than 4 nucleotides or greater than 12 nucleotides in length. In certain embodiments, the single-stranded region is 6 or 7 nucleotides long.

In some embodiments, the sd-RNA molecule has increased stability. In certain instances, the half-life of the chemically modified sd-RNA or sd-rxRNA molecule in culture medium is longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or more than 24 hours, including any intermediate values. In some embodiments, the sd-rxRNA has a half-life in culture medium of greater than 12 hours.

In some embodiments, sd-RNA is optimized to increase potency and/or reduce toxicity. In some embodiments, the nucleotide length of the guide strand and/or the follower strand and/or the number of phosphorothioate modifications in the guide strand and/or the follower strand may affect the potency of the RNA molecule in some respects, while the replacement of the 2 '-fluoro (2' F) modification with the 2 '-O-methyl (2' OMe) modification may affect the toxicity of the molecule in some respects. In some embodiments, a reduction in the amount of 2' F in the molecule is expected to reduce the toxicity of the molecule. In some embodiments, the number of phosphorothioate modifications in an RNA molecule can affect the efficiency with which the molecule is taken up into a cell, e.g., the efficiency with which the molecule is passively taken up into a cell. In some embodiments, sd-RNA is free of 2' F modifications, but is equally effective in cellular uptake and tissue penetration.

In some embodiments, the guide strand is about 18 to 19 nucleotides in length and has about 2-14 phosphate modifications. For example, the guide strand may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more than 14 phosphate modified nucleotides. The guide chain may comprise more than one modification that confers increased stability without interfering with RISC entries. Phosphate modified nucleotides, such as phosphorothioate modified nucleotides, may be located at the 3 'end, the 5' end, or throughout the guide strand. In some embodiments, the 10 nucleotides at the 3' terminus of the guide strand comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphorothioate modified nucleotides. The guide strand may also comprise 2'F and/or 2' OMe modifications, which may be located throughout the molecule. In some embodiments, the nucleotide at the first position of the guide strand (the nucleotide at the most 5 'position of the guide strand) is 2' OMe modified and/or phosphorylated. The C and U nucleotides within the guide strand may be 2' F modified. For example, the C and U nucleotides at positions 2-10 of a 19nt guide strand (or at corresponding positions in guide strands of different lengths) may be 2' F modified. The C and U nucleotides within the guide strand may also be 2' OMe modified. For example, the C and U nucleotides at positions 11-18 of a 19nt guide strand (or corresponding positions in guide strands of different lengths) may be 2' OMe modified. In some embodiments, the nucleotide at the most 3' end of the guide strand is unmodified. In certain embodiments, most of the C and U within the guide strand are 2'F modified, and the 5' end of the guide strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are 2'OMe modified and the 5' end of the guide strand is phosphorylated. In other embodiments, the C or U at positions 1 and 11-18 are 2' OMe modified, the 5' terminus of the guide strand is phosphorylated, and the C or U at positions 2-10 are 2' F modified.

delivery of sd-RNA

Self-delivering RNAi technology provides a method for direct transfection of cells with RNAi agents without the need for additional agents or techniques. The ability to transfect difficult to transfect cell lines, high in vivo activity and ease of use are features of compositions and methods that have significant functional advantages over traditional siRNA-based techniques, and thus, the sd-RNA approach has been employed in some embodiments involving methods of reducing target gene expression in the TILs of the invention. The sd-RNAi approach can deliver chemically synthesized compounds directly into a variety of primary cells and tissues in vivo and in vitro. sd-RNA described in some embodiments of the invention is available from Advirna LLC (Worcester, MA, usa).

The general structure of sd-RNA molecules is shown in FIG. 36. sd-RNA is formed as a hydrophobically modified siRNA-antisense oligonucleotide hybrid structure disclosed in, e.g., Byrne et al, 12 months 2013, j. ocular Pharmacology and therapeutics, 29 (10): 855, 864, the contents of which are incorporated herein by reference.

In some embodiments, sd-RNA oligonucleotides can be delivered to TILs described herein using sterile electroporation.

In some embodiments, the oligonucleotide may be delivered to the cell in conjunction with a transmembrane delivery system. In some embodiments, the transmembrane delivery system comprises a lipid, a viral vector, or the like. In some embodiments, the oligonucleotide agent is a self-delivering RNAi agent without any delivery agent.

In embodiments, different methods can be used to introduce an oligonucleotide (e.g., an RNA or sd-RNA as described herein) into a target cell, e.g., a nucleic acid molecule that can be expressed in a nucleic acid moleculeCommercially available methods include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Colon, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Mass.), Neon^TMTransfection System (commercially available from ThermoFisher Scientific, Waltherm, Mass.) and/or Gene Pulser II (BioRad, Denver, Colorado), Multipotor (Eppendort, Hamburg, Germany), cationic liposome-mediated Transfection using lipofection, Polymer encapsulation, peptide-mediated Transfection, or biolistic particle delivery systems, such as "Gene gun" (see, e.g., Nishikawa et al, Hum Gene Ther., 12 (8): 861-70(2001), the entire contents of which are incorporated herein by referenceHandbook II (available at world Wide Web http:// icob. sinica. edu. tw/pubweb/bio-chem/Core% 20 Facilities/Data/R401-Core/Nuclear of operator _ Manual _ II _ Apr06. pdf).

In some embodiments, the Amaxa nuclofecter.tm. -II electroporation may be performed according to manufacturer's recommendations. In some embodiments, TIL may be transfected using nuceofecto.tm. -II solution V and a set of recommended protocols for electroporation. In some embodiments, TIL may be transfected using solutions V, T and R and different electroporation protocols. In some embodiments, TIL may be transfected using a T cell nuclofeitor.tm. -II solution and different electroporation protocols. Alternative methods of nucleic acid delivery may also be used to transfect the oligonucleotides described herein: lipofectin or LIPOFECTAMIN (Invitrogen) was used for cationic liposome-mediated transfection. ECM 830(BTX) (Harvard instruments, Boston, Mass.), Gene Pulser II (BioRad, Denver, Colorado), Multiporitor (Eppendorf, Hamburg, Germany) and/or Neon may also be used ^TMElectroporation was performed by the transduction System (commercially available from ThermoFisher Scientific, Waltherm, Mass.). In some embodiments, pmaxGFP plasmid DNA (Amaxa Biosystems) can be used as a DNA control. In some embodiments, about 3, 6, 9, 12, 15 and/or after transfection may be performedTransfection Efficiency (ET) was determined by Fluorescence Activated Cell Sorting (FACS) at 18 hours. In some experiments, transfectants can be further analyzed every 12 to 24 hours until GFP control fails to detect GFP. In some embodiments, cell viability can be determined by trypan blue dye exclusion.

Oligonucleotides and oligonucleotide compositions are contacted with (e.g., contacted with, also referred to herein as administered or delivered to) and taken up by TILs described herein, including by passive uptake of the TILs. sd-RNA can be added to the TIL as described herein during the first amplification (e.g., step B), after the first amplification (e.g., during step C), before or during the second amplification (e.g., before or during step D, after step D and before the harvesting of step E, during or after the harvesting in step F, before or during the final formulation and/or transfer to an infusion bag in step F, and before any optional cryopreservation step in step F). Furthermore, sd-RNA can be added after thawing of any cryopreservation step in step F. In one embodiment, one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to cell culture media comprising TIL and other agents at a concentration of 100nM to 20mM, 200nM to 10mM, 500nM to 1mM, 1 μ Μ to 100 μ Μ, and 1 μ Μ to 100 μ Μ. In one embodiment, one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu. Msd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium. In one embodiment, more than one sd-RNA targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the pre-REP or REP stage. In one embodiment, one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: mu.g of Msd-RNA/10,000 TIL/100. mu.L of medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L of medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L of medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L of medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L of medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L of medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L of medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L of medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L of medium. In one embodiment, more than one sd-RNA targeting a gene described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the first amplification, second amplification, and/or additional amplification stages.

An oligonucleotide composition of the invention comprising sd-RNA can be contacted with the TIL during amplification as described herein, for example, by dissolving a high concentration of sd-RNA in the cell culture medium and allowing sufficient time for passive uptake. In some embodiments, a high concentration comprises 0.1 μ M sd-RNA/10,000TIL, 0.5 μ M sd-RNA/10,000TIL, 0.75 μ Msd-RNA/10,000TIL, 1 μ M sd-RNA/10,000TIL, 1.25 μ M sd-RNA/10,000TIL, 1.5 μ M sd-RNA/10,000TIL, 2 μ M sd-RNA/10,000TIL, 5 μ M sd-RNA/10,000TIL, or 10 μ M sd-RNA/10,000 TIL. In some embodiments, a high concentration comprises 2 μ M sd-RNA/10,000TIL, 5 μ M sd-RNA/10,000TIL, or 10 μ M sd-RNA/10,000 TIL. In some embodiments, a high concentration comprises 5 μ M sd-RNA/10,000TIL or up to 10 μ M sd-RNA/10,000 TIL.

In some embodiments, delivery of the oligonucleotide into the cell can be enhanced by suitable art-recognized methods (including calcium phosphate, DMSO, glycerol, or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes) using methods known in the art (see, e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No. 4,897,355; Bergan et al, Nucleic Acids Research 1993, 21: 3567).

sd-RNA combinations

In some embodiments, more than one sd-RNA is used to reduce expression of the target gene. In some embodiments, sd-RNAs targeting PD-1, TIM-3, CBLB, LAG3, and/or CISH are used together. In some embodiments, PD-1sd-RNA is used with one or more of TIM-3, CBLB, LAG3, and/or CISH to reduce expression of one or more gene targets. In some embodiments, LAG3 sd-RNA is used in combination with CISH-targeting sd-RNA to reduce gene expression of both targets. In some embodiments, sd-RNA targeting more than one of PD-1, TIM-3, CBLB, LAG3 and/or CISH herein is commercially available from Advirna LLC (Worcester, MA, USA). In some embodiments, the sd-RNA that targets one or more of PD-1, TIM-3, CBLB, LAG3, and/or CISH has the structure shown in figure 36 or figure 37.

In some embodiments, the sd-RNA targets a gene selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sd-RNA targets a gene selected from PD-1, LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, one sd-RNA targets PD-1 and another sd-RNA targets a gene selected from LAG3, TIM3, CTLA-4, TIGIT, CISH, TGF β R2, PKA, CBLB, BAFF (BR3), and combinations thereof. In some embodiments, the sd-RNA targets a gene selected from PD-1, LAG-3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, the sd-RNA targets a gene selected from PD-1 and LAG3, CISH, CBLB, TIM3, and combinations thereof. In some embodiments, one sd-RNA targets PD-1 and one sd-RNA targets LAG 3. In some embodiments, one sd-RNA targets PD-1 and one sd-RNA targets CISH. In some embodiments, one sd-RNA targets PD-1 and one sd-RNA targets CBLB. In some embodiments, one sd-RNA targets LAG3 and one sd-RNA targets CISH. In some embodiments, one sd-RNA targets LAG3 and one sd-RNA targets CBLB. In some embodiments, one sd-RNA targets CISH and one sd-RNA targets CBLB. In some embodiments, one sd-RNA targets TIM3 and one sd-RNA targets PD-1. In some embodiments, one sd-RNA targets TIM3 and one sd-RNA targets LAG 3. In some embodiments, one sd-RNA targets TIM3 and one sd-RNA targets CISH. In some embodiments, one sd-RNA targets TIM3 and one sd-RNA targets CBLB.

f. Co-stimulatory receptor or adhesion molecule overexpression

According to other embodiments, altering protein expression of TILs during a TIL amplification method may also allow for enhanced expression of more than one immune checkpoint gene in at least a portion of a therapeutic TIL population. For example, altering protein expression may result in increased expression of a stimulatory receptor, meaning that the stimulatory receptor is overexpressed compared to the expression of the stimulatory receptor without genetic modification. Non-limiting examples of immune checkpoint genes that may exhibit enhanced expression through transient alteration of protein expression in the TILs of the invention include certain chemokine receptors and interleukins, such as CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, IL-2, IL-4, IL-7, IL-10, IL-15, IL-21, NOTCH 1/2 intracellular domain (ICD) and/or NOTCH ligand mDLL 1.

(i) CCR and CCL

For adoptive T cell immunotherapy to be effective, it is necessary to properly transport T cells into tumors by chemokines. Matching between chemokines secreted by tumor cells, chemokines present in the periphery, and chemokine receptors expressed by T cells is critical for successful trafficking of T cells to the tumor bed.

According to particular embodiments, the methods of altering protein expression of the present invention may be used to increase the expression of certain chemokine receptors in TIL, such as one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and/or CX3CR 1. After adoptive transfer, overexpression of CCR may help promote effector function and proliferation of TIL. In some embodiments, the methods of the invention for altering protein expression can be used to increase the expression of CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1, CXCL8, CCL22, and/or CCL17 in TIL.

According to particular embodiments, the compositions and methods of the present invention enhance the expression of more than one of CCR2, CCR4, CCR5, CXCR2, CXCR3, and/or CX3CR1 in TIL. For example, a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population can be performed according to any of the embodiments of the methods described herein (e.g., the methods shown in process 2A or figures 20 and 21), wherein the method comprises genetically editing at least a portion of the TILs by enhancing expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, and/or CX3CR 1. As described in more detail below, the gene editing process may include the use of programmable nucleases that mediate the production of double-stranded or single-stranded breaks in chemokine receptor genes. For example, CRISPR methods, TALE methods, or zinc finger methods can be used to enhance expression of certain chemokine receptors in TILs.

In one embodiment, CCR4 and/or CCR5 adhesion molecules are inserted into the TIL population using the γ -retroviral or lentiviral methods described herein. In one embodiment, CXCR2 adhesion molecules are inserted into TIL populations using the gamma-retrovirus or lentivirus approach described by Forget et al (Frontiers Immunology2017,8,908 or Peng et al, clin. cancer res.2010,16,5458 (the disclosure of which is incorporated herein by reference)).

In some embodiments, the present invention provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(i) obtaining a first TIL population from a resected tumor of a patient;

(ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population;

(iv) exposing the second and/or third TIL populations to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression; wherein TF and/or other molecule capable of transiently altering protein expression provides for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population; wherein the alteration in expression is an increase in expression of one or more of CCR2, CCR4, CCR5, CXCR2, CXCR3, CX3CR1, CCL2(MCP-1), CCL3(MIP-1 α), CCL4(MIP1- β), CCL5(RANTES), CXCL1, CXCL8 and/or CCL 22.

(ii) Interleukins and others

According to additional embodiments, the gene editing methods of the invention can be used to increase the expression of certain interleukins (e.g., one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21) as well as the NOTCH 1/2 intracellular domain (ICD). Certain interleukins have been shown to enhance effector function of T cells and to modulate tumour control.

According to certain embodiments, the compositions and methods of the invention enhance the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21, as well as the NOTCH 1/2 intracellular domain (ICD) in the TIL. In some embodiments, the present invention provides a method of expanding Tumor Infiltrating Lymphocytes (TILs) into a therapeutic TIL population, the method comprising:

(i) obtaining a first TIL population from a resected tumor of a patient;

(ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population;

(iv) Exposing the second and/or third TIL populations to Transcription Factor (TF) and/or other molecule capable of transiently altering protein expression; wherein TF and/or other molecule capable of transiently altering protein expression provides for alteration of tumor antigen expression and/or alteration of the number of tumor antigen-specific T cells in the therapeutic TIL population; wherein the alteration in expression is an increase in the expression of one or more of IL-2, IL-4, IL-7, IL-10, IL-15, and IL-21, and the NOTCH 1/2 intracellular domain (ICD).

Til production process

Fig. 1 depicts an exemplary TIL process (referred to as process 2A) that includes some of these features, and fig. 2 depicts some of the advantages of the present embodiment of the invention relative to process 1C, as shown in fig. 13 and 14. Fig. 3 shows process 1C for comparison. Fig. 4 (higher cell number) and fig. 5 (lower cell number) show two alternative timelines for TIL treatment based on procedure 2A. Fig. 6 and 8 show an embodiment of process 2A. Fig. 13 and 14 also provide an exemplary 2A process as compared to an exemplary 1C process.

As described herein, the present invention may include steps related to the re-stimulation of cryopreserved TILs to increase their metabolic activity and thus relative health prior to transplantation into a patient, as well as methods of detecting such metabolic health. As generally outlined herein, TILs are typically taken from a patient sample and manipulated to expand their quantity prior to transplantation into the patient. In some embodiments, the TIL may optionally be genetically manipulated as described below.

In some embodiments, the TIL may be cryopreserved. Once thawed, they may also be restimulated to increase their metabolism prior to infusion into a patient.

In some embodiments, as described in detail below and in the examples and figures, the first amplification (including the process referred to as pre-REP and the process shown as step a in fig. 8) is reduced to 3 days to 14 days, and the second amplification (including the process referred to as REP and the process shown as step B in fig. 8) is reduced to 7 days to 14 days. In some embodiments, as described in the examples and as shown in fig. 4, 5, 6, and 7, the first amplification (e.g., the amplification described in step B in fig. 8) is reduced to 11 days and the second amplification (e.g., the amplification described in step D in fig. 8) is reduced to 11 days. In some embodiments, as described in detail below and in the examples and figures, the sum of the first and second amplifications (e.g., the amplifications described in step B and step D in fig. 8) is reduced to 22 days.

The "step" of reference A, B, C et al below refers to FIG. 8 and certain embodiments described herein. The order of the steps described below and in fig. 8 is exemplary, and any combination or order of steps, as well as additional steps, repetition of steps, and/or omission of steps is contemplated by the present application and the methods disclosed herein.

A. Step A: obtaining a patient tumor sample

Typically, TILs are initially obtained from a patient tumor sample ("primary TILs") and then expanded into larger populations for further manipulation as described herein, optionally cryopreserved, restimulated as described herein, and optionally evaluated for phenotypic and metabolic parameters as indicators of TIL health.

Patient tumor samples can be obtained using methods known in the art, typically by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and TIL cells. In general, the tumor sample may be from any solid tumor, including a primary tumor, an invasive tumor, or a metastatic tumor. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematologic malignancy. The solid tumor can be any cancer type, including but not limited to breast cancer, pancreatic cancer, prostate cancer, colorectal cancer, lung cancer, brain cancer, kidney cancer, stomach cancer, and skin cancer (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, useful TILs are obtained from malignant melanoma tumors, as they are reported to have particularly high levels of TIL.

The term "solid tumor" refers to an abnormal tissue mass that generally does not contain cysts or fluid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to malignant, neoplastic, or cancerous solid tumors. Solid tumor cancers include, but are not limited to, sarcomas, carcinoma of the epithelium (carcinoma), and lymphomas, such as lung, breast, triple negative breast, prostate, colon, rectal, and bladder cancers. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, Head and Neck Squamous Cell Carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple negative breast cancer, and non-small cell lung cancer. The tissue structure of a solid tumor comprises interdependent tissue compartments including parenchymal (cancer cells) and supporting stromal cells (into which cancer cells are dispersed and which may provide a supporting microenvironment).

The term "hematologic malignancy" refers to cancers and tumors of mammalian hematopoietic and lymphoid tissues, including but not limited to blood, bone marrow, lymph nodes, and tissues of the lymphatic system. Hematological malignancies are also known as "liquid tumors". Hematological malignancies include, but are not limited to: acute Lymphocytic Leukemia (ALL), Chronic Lymphocytic Lymphoma (CLL), Small Lymphocytic Lymphoma (SLL), Acute Myelogenous Leukemia (AML), Chronic Myelogenous Leukemia (CML), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphoma. The term "B cell hematologic malignancy" refers to a hematologic malignancy that affects B cells.

Once obtained, tumor samples are broken into 1mm, typically using sharp dissection³To about 8mm³Of a small piece of about 2mm³To 3mm³Is particularly useful. TILs were cultured from these fragments using enzymatic tumor digests. Such tumor digests can be produced by incubation in enzyme culture medium (e.g., RPMI, Roswell Park Memorial Institute)1640 buffer, 2mM glutamic acid, 10mcg/mL gentamicin, 30U/mL DNase, and 1.0mg/mL collagenase followed by mechanical dissociation (e.g., using a tissue dissociator). The tumor can be isolated mechanically by placing the tumor in an enzyme medium for about 1 minute, followed by 5% CO ₂Incubation at 37 ℃ for 30 minutes, then repeated mechanical dissociation and incubation under the above conditions until only small tissue fragments are present, thereby producing tumor digests. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, the FICOLL branched-chain hydrophilic polysaccharide can be used to perform density gradient separation to remove these cells. Alternative methods known in the art may be used, such as those described in U.S. patent application publication No. 2012/0244133a1, the disclosure of which is incorporated by referenceHerein incorporated. Any of the foregoing methods may be used in any embodiment of the methods for expanding TILs or treating cancer described herein.

Typically, the harvested cell suspension is referred to as a "primary cell population" or a "freshly harvested" cell population.

In some embodiments, disruption includes physical disruption, including, for example, dissection and digestion. In some embodiments, the disruption is physical disruption. In some embodiments, the disruption is dissection. In some embodiments, the disruption is by digestion. In some embodiments, TILs may be initially cultured from enzymatic tumor digests and tumor fragments obtained from a patient. In one embodiment, the TIL may be initially cultured from an enzymatic tumor digest and tumor debris obtained from the patient.

In some embodiments, when the tumor is a solid tumor, the tumor is physically disrupted, e.g., after obtaining the tumor sample in step a (as shown in fig. 8). In some embodiments, the disruption occurs prior to cryopreservation. In some embodiments, the disruption occurs after cryopreservation. In some embodiments, disruption occurs after obtaining the tumor and without any cryopreservation. In some embodiments, the tumor is disrupted and 10, 20, 30, 40 or more fragments or pieces are placed in each container for first expansion. In some embodiments, the tumor is broken up and 30 or 40 pieces or bits are placed in each container for the first expansion. In some embodiments, the tumor is broken and 40 pieces or bits are placed in each container for the first expansion. In some embodiments, the plurality of fragments comprises about 4 to about 50 fragments, wherein each fragment has a volume of about 27mm³. In some embodiments, the plurality of fragments comprises about 30 to about 60 fragments, having a total volume of about 1300mm³To about 1500mm³. In some embodiments, the plurality of fragments comprises about 50 fragments having a total volume of about 1350mm ³. In some embodiments, the plurality of fragments comprises about 50 fragments and has a total mass of about 1 gram to about 1.5 grams. In some embodiments, the plurality of fragments comprises about 4 fragments.

In some casesIn embodiments, the TIL is obtained from a tumor fragment. In some embodiments, the tumor fragments are obtained by sharp dissection. In some embodiments, the tumor fragments are about 1mm³To 10mm³. In some embodiments, the tumor fragments are about 1mm³To 8mm³. In some embodiments, the tumor fragments are about 1mm³. In some embodiments, the tumor fragments are about 2mm³. In some embodiments, the tumor fragment is about 3mm³. In some embodiments, the tumor fragments are about 4mm³. In some embodiments, the tumor fragments are about 5mm³. In some embodiments, the tumor fragments are about 6mm³. In some embodiments, the tumor fragments are about 7mm³. In some embodiments, the tumor fragments are about 8mm³. In some embodiments, the tumor fragment is about 9mm³. In some embodiments, the tumor fragments are about 10mm³. In some embodiments, the tumor is 1mm to 4mm x 1mm to 4 mm. In some embodiments, the tumor is 1mm x 1 mm. In some embodiments, the tumor is 2mm x 2 mm. In some embodiments, the tumor is 3mm x 3 mm. In some embodiments, the tumor is 4mm x 4 mm.

In some embodiments, the tumor is resected to minimize the amount of bleeding tissue, necrotic tissue, and/or adipose tissue on each nub. In some embodiments, the tumor is resected to minimize the amount of bleeding tissue on each patch. In some embodiments, the tumor is resected to minimize the amount of necrotic tissue on each nub. In some embodiments, the tumor is resected to minimize the amount of adipose tissue on each nub.

In some embodiments, tumor disruption is performed in order to maintain tumor internal structure. In some embodiments, tumor disruption is performed without a scalpel performing a sawing motion. In some embodiments, the TIL is obtained from a tumor digest. In some embodiments, by incubation in an enzyme medium (such as, but not limited to, RPMI 1640, 2mm GlutaMAX, 10mg/mL gentamicin, 30U/mL DNase, and 1.0mg/mL collagenase),mechanical dissociation (GentlemACS, Miltenyi Biotec, Aubum, Calif.) was then performed to generate tumor digests. After the tumor is placed in the enzyme medium, the tumor can be mechanically dissociated for about 1 minute. The solution was then incubated at 37 ℃ with 5% CO₂Incubated for 30 minutes and then mechanically disrupted again for about 1 minute. At 37 deg.C, 5% CO ₂After 30 minutes of incubation again, the tumor can be mechanically disrupted for a third time for about 1 minute. In some embodiments, after the third mechanical disruption, if large pieces of tissue are present, the sample is subjected to 1 or 2 additional mechanical dissociation, with or without 5% CO at 37 ℃₂The following additional 30 minutes incubation. In some embodiments, at the end of the final incubation, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation can be performed using Ficoll to remove these cells.

In some embodiments, the cell suspension harvested prior to the first expansion step is referred to as a "primary cell population" or a "freshly harvested" cell population.

In some embodiments, the cells may optionally be frozen after harvesting the sample and cryopreserved prior to expansion as described in step B, which is described in further detail below and illustrated in fig. 8.

B. And B: first amplification

In some embodiments, the methods of the invention provide for obtaining a young TIL that is capable of increasing the replication cycle after administration to a subject/patient relative to an older TIL (i.e., a TIL that has further undergone more rounds of replication prior to administration to the subject/patient), and thus may provide additional therapeutic benefits. Features of young TILs have been described in the literature, such as Donia et al, Scandinavian journal of Immunology, 75: 157-167 (2012); dudley et al, Clin cancer Res, 16: 6122-6131 (2010); huang et al, J immunoher, 28 (3): 258-267 (2005); besser et al, Clincancer Res, 19 (17): OF1-OF9 (2013); besser et al, J Implanter, 32: 415-423 (2009); bunds et al, J immunoher, 32: 415-423 (2009); robbins et al, J Immunol, 2004; 173: 7125-7130; shen et al, J immunoher, 30: 123-129 (2007); zhou et al, J immunoher, 28: 53-62(2005) and Tran et al, JImmunether, 31: 742-751(2008), the entire contents of which are incorporated herein by reference in their entirety.

Multiple antigen receptors for T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene fragments: v (variable), D (variable), J (connecting), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T Cell Receptors (TCRs). The present invention provides methods of producing TILs that demonstrate and increase the diversity of T cell repertoires (T-cell reporters). In some embodiments, the TILs obtained by the present methods exhibit increased diversity in the T cell repertoire. In some embodiments, TILs obtained by the methods of the invention exhibit increased diversity in T cell banks compared to freshly harvested TILs and/or TILs prepared using methods other than those provided herein, including, for example, methods other than those shown in fig. 8. In some embodiments, the TIL obtained by the present methods exhibits increased diversity in the T cell bank as compared to freshly harvested TIL and/or TIL prepared using a method referred to as process 1C as shown in fig. 13. In some embodiments, the TIL obtained from the first expansion exhibits an increase in diversity of the T cell pool. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, the immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, the diversity is T cell receptor diversity. In some embodiments, the diversity is a diversity of T cell receptors selected from one of alpha, beta, gamma, and receptor. In some embodiments, expression of T Cell Receptors (TCR) alpha and/or beta is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, expression of TCRab (i.e., TCR α/β) is increased.

After dissection or digestion of the tumor fragments (e.g., as described in step a of fig. 8), under conditions that favor TIL growth over tumors and other cells,the resulting cells were cultured in serum containing IL-2. In some embodiments, the tumor digest is incubated in a medium comprising inactivated human AB serum and 6000IU/mL IL-2 in 2mL wells. Culturing the primary cell population for a period of time, typically 3 to 14 days, produces a large population of TILs, typically about 1X 10⁸A plurality of TIL cells. In some embodiments, this primary cell population is cultured for 7 to 14 days, resulting in a large population of TILs, typically about 1 × 10⁸A plurality of TIL cells. In some embodiments, this primary cell population is cultured for 10 to 14 days to produce a large population of TILs, typically about 1 × 10⁸A plurality of TIL cells. In some embodiments, this population of primary cells is cultured for about 11 days, resulting in a large population of TILs, typically about 1 × 10⁸A plurality of TIL cells.

In a preferred embodiment, amplification of the TIL may be performed as follows: an initial bulk TIL amplification step (e.g., as described in step B of fig. 8, which may include a process referred to as pre-REP) is used as described below and herein, followed by a second amplification (step D, including a process referred to as a rapid amplification protocol (REP) step) as described below and herein, optionally followed by cryopreservation, followed by a second step D (including a process referred to as a restimulation REP step) as described below and herein. Alternatively, the TIL obtained from this process may be subjected to characterization of phenotypic and metabolic parameters as described herein.

In embodiments where TIL culture is initiated in 24-well plates, for example, using Costar 24-well cell culture plates, flat bottom (Corning Inc., Corning, N.Y.), 1X 10 wells in 2mL Complete Medium (CM) containing IL-2 (6000 IU/mL; Chiron Corp., Emeryville, Calif.) can be seeded in each well⁶One tumor digesting cell or one tumor fragment. In some embodiments, the tumor fragment is about 1mm³To 10mm³。

In some embodiments, the first amplification medium is referred to as "CM", where CM is an abbreviation for medium. In some embodiments, the CM of step B consists of RPMI 1640 with GlutaMAX supplemented with 10% human AB serum, 25mm hepes and 10mg/mL gentamicin. The volume of the solution is 40mL and the solution is transparentThe gas silicon bottom is 10cm²In embodiments where culture was started in gas permeable flasks (e.g., G-Rex 10; Wilson Wolf Manufacturing, New Bladeton, MN) (FIG. 1), each flask was filled with 10X 10 in 10 to 40mL of CM containing IL-2⁶To 40X 10⁶One live tumor digesting cell or 5 to 30 tumor fragments. At 37 deg.C, 5% CO₂The G-Rex10 and 24-well plates were incubated in the humidified incubator, and 5 days after the start of the culture, half of the medium was removed and replaced with fresh CM and IL-2, and after 5 days, half of the medium was replaced every 2 to 3 days.

After preparing the tumor fragments, the resulting cells (i.e., fragments) are cultured in serum containing IL-2 under conditions that favor the growth of TIL over tumors and other cells. In some embodiments, tumor fragments are incubated in 2mL wells of medium containing inactivated human AB serum (or in some cases, as described herein, in the presence of aAPC cell populations) and 6000IU/mL IL-2. The primary cell population is cultured for a period of time, typically 10 to 14 days, to produce a large population of TILs, typically about 1X 10⁸A plurality of TIL cells. In some embodiments, the growth medium during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). In some embodiments, 1mg vial of IL-2 stock solution has a 20X 10⁶IU/mg to 30X 10⁶Specific activity IU/mg. In some embodiments, 1mg vial of IL-2 stock solution has a 20X 10⁶IU/mg to 30X 10⁶Specific activity IU/mg. In some embodiments, 1mg vial of IL-2 stock solution has a 25X 10⁶Specific activity IU/mg. In some embodiments, 1mg vial of IL-2 stock solution has a 30X 10⁶Specific activity IU/mg. In some embodiments, the IL-2 stock solution has a final concentration of 4X 10 ⁶IU/mg to 8X 10⁶IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 5X 10⁶IU/mg to 7X 10⁶IU/mg IL-2. In some embodiments, the IL-2 stock solution has a final concentration of 6X 10⁶IU/mg IL-2. In some embodiments, an IL-2 stock solution is prepared as described in example 4. In some embodiments, the first amplification medium comprisesAbout 10,000IU/mL IL-2, about 9,000IU/mL IL-2, about 8,000IU/mL IL-2, about 7,000IU/mL IL-2, about 6000IU/mL IL-2, or about 5,000IU/mL IL-2. In some embodiments, the first amplification medium comprises about 9,000IU/mL to about 5,000IU/mL IL-2. In some embodiments, the first amplification medium comprises about 8,000IU/mL to about 6,000 IU/mLIL-2. In some embodiments, the first amplification medium comprises about 7,000IU/mL to about 6,000IU/mL IL-2. In some embodiments, the first amplification medium comprises about 6,000IU/mL IL-2. In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, the cell culture medium comprises about 1000IU/mL, about 1500IU/mL, about 2000IU/mL, about 2500IU/mL, about 3000IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, or about 8000IU/mL IL-2. In one embodiment, the cell culture medium comprises 1000 to 2000IU/mL, 2000 to 3000IU/mL, 3000 to 4000IU/mL, 4000 to 5000IU/mL, 5000 to 6000IU/mL, 6000 to 7000IU/mL, 7000 to 8000IU/mL, or about 8000IU/mL IL-2.

In some embodiments, the first amplification medium comprises about 500IU/mL IL-15, about 400IU/mL IL-15, about 300IU/mL IL-15, about 200IU/mL IL-15, about 300IU/mL IL-15, 180IU/mL IL-15, about 160IU/mL IL-15, about 140IU/mL IL-15, about 120IU/mL IL-15, or about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 500IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 400IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 300IU/mL IL-15 to about 100IU/mL IL-15. In some embodiments, the first amplification medium comprises about 200IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180IU/mL IL-15. In one embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the first amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 20IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 15IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 10IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the first amplification medium comprises about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the first amplification medium comprises about 2IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5IU/mL IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1IU/mL IL-21.

In one embodiment, the cell culture medium comprises an OKT-3 antibody. In a preferred embodiment, the cell culture medium comprises about 30ng/mL of OKT3 antibody. In one embodiment, the cell culture medium comprises about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1 μ g/mL of OKT3 antibody. In one embodiment, the cell culture medium comprises 0.1ng/mL to 1ng/mL, 1ng/mL to 5ng/mL, 5ng/mL to 10ng/mL, 10ng/mL to 20ng/mL, 20ng/mL to 30ng/mL, 30ng/mL to 40ng/mL, 40ng/mL to 50ng/mL, 50ng/mL to 100ng/mL of the OKT3 antibody. In some embodiments, the cell culture medium does not comprise an OKT-3 antibody.

In some embodiments, the cell culture medium comprises more than one TNFRSF agonist. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of Uluzumab, Utuzumab, EU-101, a fusion protein, and fragments, derivatives, variants, biological analogs and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 0.1 μ g/mL to 100 μ g/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 20 to 40 μ g/mL in the cell culture medium.

In some embodiments, the cell culture medium comprises IL-2 at an initial concentration of about 3000IU/mL and OKT-3 antibody at an initial concentration of about 30ng/mL in addition to the one or more TNFRSF agonists, wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, the first amplification medium is referred to as "CM (abbreviation for medium)". In some embodiments, it is referred to as CM1 (medium 1). In some embodiments, CM consists of RPMI 1640 and GlutaMAX supplemented with 10% human AB serum, 25mM Hepes and 10mg/mL gentamicin. In some embodiments, the composition has a capacity of 40mL and 10cm²The culture is started in gas-permeable silicon-bottomed flasks (e.g., G-Rex 10; Wilson Wolf Manufacturing, New Brighton, MN), each containing 10mL to 40mL of CM with IL-2 added thereto, 10X 10 of the CM⁶To 40X 10⁶One live tumor digesting cell or 5 to 30 tumor fragments. Both G-Rex10 and 24 well plates were incubated in a humidified incubator at 37 ℃ with 5% CO₂Medium incubation, culturing 5 days after the start of culturing, taking out half of the medium and replacing it with fresh CM and IL-2, and after 5 days, replacing half of the medium every 2 to 3 days. In some embodiments, the CM is CM1 described in the examples, see example 5. In some embodiments, the first expansion occurs in the initial cell culture medium or the first cell culture medium. In some embodiments, the initial cell culture medium or the first cell culture medium comprises IL-2.

In some embodiments, as described in the examples and figures, the first amplification (including, for example, those described in step B of fig. 8, which may include those sometimes referred to as pre-REP) process is shortened to 3 days to 14 days. In some embodiments, as described in the examples and shown in fig. 4 and 5, and including, for example, amplification as described in step B of fig. 8, the first amplification (including, for example, those described in step B of fig. 8, which may include those sometimes referred to as pre-REP) is reduced to 7 days to 14 days. In some embodiments, the first amplification of step B is shortened to 10 to 14 days, as described in the examples and shown in fig. 4 and 5. In some embodiments, in a first example, as described in the examples and shown in fig. 4 and 5, and including amplification such as described in step B of fig. 8, the first amplification is shortened to 11 days.

In some embodiments, the first TIL amplification may be performed for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the first TIL amplification may be performed for 1 day to 14 days. In some embodiments, the first TIL amplification may be performed for 2 days to 14 days. In some embodiments, the first TIL amplification may be performed for 3 to 14 days. In some embodiments, the first TIL amplification may be performed for 4 to 14 days. In some embodiments, the first TIL amplification may be performed for 5 days to 14 days. In some embodiments, the first TIL amplification may be performed for 6 days to 14 days. In some embodiments, the first TIL amplification may be performed for 7 days to 14 days. In some embodiments, the first TIL amplification may be performed for 8 days to 14 days. In some embodiments, the first TIL amplification may be performed for 9 to 14 days. In some embodiments, the first TIL amplification may be performed for 10 to 14 days. In some embodiments, the first TIL amplification may be performed for 11 days to 14 days. In some embodiments, the first TIL amplification may be performed for 12 days to 14 days. In some embodiments, the first TIL amplification may be performed for 13 to 14 days. In some embodiments, the first TIL amplification may be performed for 14 days. In some embodiments, the first TIL amplification may be performed for 1 day to 11 days. In some embodiments, the first TIL amplification may be performed for 2 days to 11 days. In some embodiments, the first TIL amplification may be performed for 3 days to 11 days. In some embodiments, the first TIL amplification may be performed for 4 to 11 days. In some embodiments, the first TIL amplification may be performed for 5 days to 11 days. In some embodiments, the first TIL amplification may be performed for 6 days to 11 days. In some embodiments, the first TIL amplification may be performed for 7 days to 11 days. In some embodiments, the first TIL amplification may be performed for 8 to 11 days. In some embodiments, the first TIL amplification may be performed for 9 to 11 days. In some embodiments, the first TIL amplification may be performed for 10 to 11 days. In some embodiments, the first TIL amplification may be performed for 11 days.

In some embodiments, during the first amplification, a combination of IL-2, IL-7, IL-15 and/or IL-21 is employed as a combination. In some embodiments, IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof may be included during the first amplification, including, for example, during step B according to FIG. 8 and as described herein. In some embodiments, during the first amplification, a combination of IL-2, IL-15 and IL-21 is employed as the combination. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step B according to FIG. 8 and as described herein.

In some embodiments, as described in the examples and shown in FIGS. 4 and 5, the first amplification (including the process referred to as pre-REP; e.g., according to step B of FIG. 8) process is shortened to 3 days to 14 days. In some embodiments, the first amplification of step B is shortened to 7 to 14 days, as described in the examples and shown in fig. 4 and 5. In some embodiments, the first amplification of step B is shortened to 10 to 14 days, as described in the examples and shown in fig. 4, 5, 6 and 7. In some embodiments, the first amplification is reduced to 11 days as described in the examples and shown in fig. 4, 5, 6, and 7.

In some embodiments, the first amplification (e.g., according to step B of fig. 8) is performed in a closed system bioreactor. In some embodiments, the TIL amplification is performed using a closed system, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, for example, the single bioreactor employed is G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, during the first amplification (e.g., according to step B of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu. Msd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium. In some embodiments, more than one sd-RNA targeting a gene described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the first amplification (e.g., according to step B of fig. 8). In one embodiment, during the first amplification (e.g., according to step B of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: mu.M sd-RNA/10,000TIL, 0.5. mu.M sd-RNA/10,000TIL, 0.75. mu.M sd-RNA/10,000TIL, 1. mu.M sd-RNA/10,000TIL, 1.25. mu.M sd-RNA/10,000TIL, 1.5. mu.M sd-RNA/10,000TIL, 2. mu.M sd-RNA/10,000TIL, 5. mu.M sd-RNA/10,000TIL, or 10. mu.M sd-RNA/10,000 TIL. In one embodiment, during the first amplification (e.g., according to step B of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

C. And C: transition from first amplification to second amplification

In some cases, a large population of TIL obtained from the first amplification may be immediately cryopreserved using the protocol discussed below, including, for example, the TIL population obtained from step B shown in fig. 8. Alternatively, the TIL population harvested from the first amplification (referred to as a second TIL population) may be subjected to a second amplification (which may include amplification sometimes referred to as REP) and then cryopreserved as described below. Similarly, where a genetically modified TIL is to be used in therapy, either the first TIL population (sometimes referred to as a bulk TIL population) or the second TIL population (which in some embodiments may include a population referred to as a REP TIL population) may be genetically modified prior to amplification or after the first amplification and prior to the second amplification for appropriate therapy.

In some embodiments, the TIL obtained from the first amplification (e.g., step B as shown in fig. 7) is stored until phenotypic selection is performed. In some embodiments, the TIL obtained from the first amplification (e.g., step B as shown in fig. 7) is not stored but is directly subjected to a second amplification. In some embodiments, the TIL obtained from the first amplification is not cryopreserved after the first amplification and before the second amplification. In some embodiments, the transition from the first amplification to the second amplification occurs at about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between about 3 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between about 4 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between about 4 days and 10 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between about 7 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs about 14 days from the time the fragmentation occurs.

In some embodiments, the transition from the first amplification to the second amplification occurs at about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 1 day to 14 days from the time the fragmentation occurs. In some embodiments, the first TIL amplification may be performed for 2 days to 14 days. In some embodiments, the transition from the first amplification to the second amplification occurs between 3 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 4 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 5 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 6 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 7 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 8 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 9 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 10 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 11 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 12 days to 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 13 days and 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 14 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs from 1 day to 11 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 2 days and 11 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 3 days and 11 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 8 days and 11 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 9 days and 11 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs between 10 days and 11 days from the time the fragmentation occurs. In some embodiments, the transition from the first amplification to the second amplification occurs 11 days from the time the fragmentation occurs.

In some embodiments, the TIL is not stored after the first amplification and before the second amplification, and the TIL is directly subjected to the second amplification (e.g., in some embodiments, as shown in fig. 8, no storage is performed during the transition from step B to step D). In some embodiments, the transition occurs in a closed system, as described herein. In some embodiments, the TILs from the first amplification (i.e., the second TIL population) are passed directly to the second amplification without undergoing a transition period.

In some embodiments, the transition from the first amplification to the second amplification is performed in a closed system bioreactor (e.g., according to step C of fig. 8). In some embodiments, the TIL amplification is performed using a closed system, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, for example, the single bioreactor employed is G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, during the transition from the first amplification to the second amplification (e.g., according to step C of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other reagents in amounts selected from: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium. In some embodiments, more than one sd-RNA targeting a gene described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the transition from the first amplification to the second amplification (e.g., according to step C of fig. 8). In one embodiment, during the transition from the first amplification to the second amplification (e.g., according to step C of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other reagents in amounts selected from: mu.M sd-RNA/10,000TIL, 0.5. mu.M sd-RNA/10,000TIL, 0.75. mu.M sd-RNA/10,000TIL, 1. mu.M sd-RNA/10,000TIL, 1.25. mu.M sd-RNA/10,000TIL, 1.5. mu.M sd-RNA/10,000TIL, 2. mu.M sd-RNA/10,000TIL, 5. mu.M sd-RNA/10,000TIL, or 10. mu.M sd-RNA/10,000 TIL. In some embodiments, more than one sd-RNA targeting a gene described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the transition from the first amplification to the second amplification (e.g., according to step C of fig. 8).

1. Cytokine

As known in the art, the amplification methods described herein typically use media with high doses of cytokines (especially IL-2).

Alternatively, rapid expansion and/or secondary expansion of TIL may additionally be performed using a combination of cytokines; wherein combinations of two or more of IL-2, IL-15 and IL-21 are as outlined in International publication Nos. WO2015/189356 and WO2015/189357, the entire contents of which are expressly incorporated herein by reference. Thus, possible combinations include IL-2 and IL-15, IL-2 and IL-21, IL-15 and IL-21, and IL-2, IL-15 and IL-21, the latter of which has been found to be particularly useful in many embodiments. The use of a combination of cytokines is particularly advantageous for the production of lymphocytes, particularly the T cells described herein.

D. Step D: second amplification

In some embodiments, the number of TIL cell populations increases after harvesting and initial bulk processing, e.g., after steps a and B, this transition is referred to as step C, as shown in fig. 8. Such further amplification, referred to herein as a second amplification, may comprise an amplification process commonly referred to in the art as a rapid amplification process (REP; and the process shown in step D of FIG. 8). The second expansion is typically accomplished in a gas permeable container using a medium comprising a plurality of components including feeder cells, a cytokine source, and an anti-CD 3 antibody.

In some embodiments, the second amplification or second TIL amplification (which may include amplification sometimes referred to as REP; and the process shown in step D of FIG. 8) may be performed using any TIL flask or vessel known to those skilled in the art. In some embodiments, the second TIL amplification may be performed for 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the second TIL amplification may be performed for about 7 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 8 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 9 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 10 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 11 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 12 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 13 days to about 14 days. In some embodiments, the second TIL amplification may be performed for about 14 days.

In one embodiment, a second amplification can be performed in a gas permeable container using the methods of the present disclosure (including, for example, amplification referred to as REP; and the process as shown in step D of FIG. 7). For example, TIL can be rapidly expanded using non-specific T cell receptor stimulation in the presence of interleukin 2(IL-2) or interleukin-15 (IL-15). Non-specific T cell receptor stimulation may include, for example, an anti-CD 3 antibody, such as OKT3 at about 30ng/mL, a mouse monoclonal anti-CD 3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Auburn, Calif.) or UHCT-1 (commercially available from BioLegend, San Diego, Calif., U.S.A.). During the second expansion, TIL may be rapidly expanded by inducing further stimulation of TIL in vitro with a vector comprising more than one cancer antigen (including antigenic portions, e.g., epitopes, thereof), which may optionally be expressed by a vector, such as a human leukocyte antigen A2(HLA-A2) binding peptide, e.g., 0.3 μ M MART-L26-35(27L) or gp 100: 209-217 (210M). Other suitable antigens may include, for example, NY-ESO-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. TIL can also be rapidly expanded by restimulation with the same cancer antigen pulsed with antigen presenting cells expressing HLA-A2. Alternatively, TIL may be further restimulated, for example, with irradiated autologous lymphocytes or with irradiated HLA-A2+ allogenic lymphocytes and IL-2. In some embodiments, the restimulation occurs as part of a second amplification. In some embodiments, the second expansion occurs in the presence of irradiated autologous lymphocytes or irradiated HLA-a2+ allogenic lymphocytes and IL-2.

In one embodiment, the cell culture medium further comprises IL-2. In some embodiments, the cell culture medium comprises about 3000IU/mL IL-2. In one embodiment, the cell culture medium comprises about 1000IU/mL, about 1500IU/mL, about 2000IU/mL, about 2500IU/mL, about 3000IU/mL, about 3500IU/mL, about 4000IU/mL, about 4500IU/mL, about 5000IU/mL, about 5500IU/mL, about 6000IU/mL, about 6500IU/mL, about 7000IU/mL, about 7500IU/mL, or about 8000IU/mL IL-2. In one embodiment, the cell culture medium comprises 1000 to 2000IU/mL, 2000 to 3000IU/mL, 3000 to 4000IU/mL, 4000 to 5000IU/mL, 5000 to 6000IU/mL, 6000 to 7000IU/mL, 7000 to 8000IU/mL, or 8000IU/mL IL-2.

In one embodiment, the cell culture medium comprises OKT3 antibody. In some embodiments, the cell culture medium comprises about 30ng/mL of OKT3 antibody. In one embodiment, the cell culture medium comprises about 0.1ng/mL, about 0.5ng/mL, about 1ng/mL, about 2.5ng/mL, about 5ng/mL, about 7.5ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 50ng/mL, about 60ng/mL, about 70ng/mL, about 80ng/mL, about 90ng/mL, about 100ng/mL, about 200ng/mL, about 500ng/mL, and about 1 μ g/mL of OKT3 antibody. In one embodiment, the cell culture medium comprises 0.1ng/mL to 1ng/mL, 1ng/mL to 5ng/mL, 5ng/mL to 10ng/mL, 10ng/mL to 20ng/mL, 20ng/mL to 30ng/mL, 30ng/mL to 40ng/mL, 40ng/mL to 50ng/mL, 50ng/mL to 100ng/mL of the OKT3 antibody. In some embodiments, the cell culture medium does not comprise an OKT-3 antibody.

In some embodiments, initiating the first expansion of the cell culture medium comprises one or more TNFRSF agonists in the cell culture medium. In some embodiments, the TNFRSF agonist comprises a 4-1BB agonist. In some embodiments, the TNFRSF agonist is a 4-1BB agonist, and the 4-1BB agonist is selected from the group consisting of: ulmacezumab, Ulvacizumab, EU-101, fusion proteins and fragments, derivatives, variants, biological analogs and combinations thereof. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 0.1 μ g/mL to 100 μ g/mL in the cell culture medium. In some embodiments, the TNFRSF agonist is added at a concentration sufficient to achieve 20 to 40 μ g/mL in the cell culture medium.

In some embodiments, the first expanded cell culture medium initiating the first expansion comprises IL-2 at an initial concentration of about 3000IU/mL and OKT-3 antibody at an initial concentration of about 30IU/mL in addition to the one or more TNFRSF agonists, wherein the one or more TNFRSF agonists comprise a 4-1BB agonist.

In some embodiments, a combination of IL-2, IL-7, IL-15, and/or IL-21 is employed as a combination during the second amplification. In some embodiments, the second amplification period (including for example according to figure 8 step D process, and as described herein) can include IL-2, IL-7, IL-15 and/or IL-21 and any combination thereof. In some embodiments, a combination of IL-2, IL-15, and IL-21 is employed as a combination during the second amplification. In some embodiments, IL-2, IL-15, and IL-21, and any combination thereof, may be included during step D according to FIG. 8, and as described herein.

In some embodiments, the second expansion can be performed in a supplemented cell culture medium comprising IL-2, OKT-3, antigen presenting feeder cells, and optionally a TNFRSF agonist. In some embodiments, the second expansion occurs in a supplemental cell culture medium. In some embodiments, the supplemented cell culture medium comprises IL-2, OKT-3, and antigen presenting feeder cells. In some embodiments, the second cell culture medium comprises IL-2, OKT-3, and antigen presenting cells (APCs; also referred to as antigen presenting feeder cells). In some embodiments, the second expansion occurs in a cell culture medium comprising IL-2, OKT-3, and antigen presenting feeder cells (i.e., antigen presenting cells).

In some embodiments, the second amplification medium comprises about 500IU/mL IL-15, about 400IU/mL IL-15, about 300IU/mL IL-15, about 200IU/mL IL-15, about 180IU/mL IL-15, about 160IU/mL IL-15, about 140IU/mL IL-15, about 120IU/mL IL-15, or about 100IU/mL IL-15. In some embodiments, the second amplification medium comprises about 500IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the second amplification medium comprises about 400IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the second amplification medium comprises about 300IU/mL of IL-15 to about 100IU/mL of IL-15. In some embodiments, the second amplification medium comprises about 200IU/mL IL-15. In some embodiments, the cell culture medium comprises about 180IU/mL IL-15. In one embodiment, the cell culture medium further comprises IL-15. In a preferred embodiment, the cell culture medium comprises about 180IU/mL IL-15.

In some embodiments, the second amplification medium comprises about 20IU/mL IL-21, about 15IU/mL IL-21, about 12IU/mL IL-21, about 10IU/mL IL-21, about 5IU/mL IL-21, about 4IU/mL IL-21, about 3IU/mL IL-21, about 2IU/mL IL-21, about 1IU/mL IL-21, or about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 20IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second amplification medium comprises about 15IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second amplification medium comprises about 12IU/mL IL-21 to about 0.5IU/mL IL-21. In some embodiments, the second amplification medium comprises about 10IU/mL of IL-21 to about 0.5IU/mL of IL-21. In some embodiments, the second amplification medium comprises about 5IU/mL IL-21 to about 1IU/mL IL-21. In some embodiments, the second amplification medium comprises about 2IU/mL IL-21. In some embodiments, the cell culture medium comprises about 1IU/mL IL-21. In some embodiments, the cell culture medium comprises about 0.5IU/mL IL-21. In one embodiment, the cell culture medium further comprises IL-21. In a preferred embodiment, the cell culture medium comprises about 1IU/mL IL-21.

In some embodiments, the antigen presenting feeder cells (APCs) are PBMCs. In one embodiment, the ratio of TIL to PBMCs and/or antigen presenting cells in the rapid expansion and/or the second expansion is about 1: 25. about 1: 50. about 1: 100. about 1: 125. about 1: 150. about 1: 175. about 1: 200. about 1: 225. about 1: 250. about 1: 275. about 1: 300. about 1: 325. about 1: 350. about 1: 375. about 1: 400, or about 1: 500. in one embodiment, the ratio of TIL to PBMC in the rapid amplification and/or the second amplification is 1: 50 to 1: 300. in one embodiment, the ratio of TIL to PBMC in the rapid amplification and/or the second amplification is 1: 100 to 1: 200.

in one embodiment, REP and/or the second amplification is performed in a flask; wherein a large amount of TIL is mixed with 100 or 200 fold excess of inactivated feeder cells, 30mg/mL of OKT3 anti-CD 3 antibody and 3000IU/mL IL-2 in 150mL of culture medium. Media replacement (typically 2/3 media replacement by aspiration of fresh media) is performed until the cells are transferred to an alternate growth chamber. As discussed more fully below, alternative growth chambers include G-REX flasks and gas permeable containers.

In some embodiments, the second amplification (which may include a process referred to as the REP process) is shortened to 7 days to 14 days, as described in the examples and figures. In some embodiments, the second amplification is shortened to 11 days.

In one embodiment, REP and/or the second expansion may be performed using a T-175 flask and gas permeable bag (Tran et al, J.I mmunither., 2008, 31, 742-51; Dudley et al, J.I mmunither., 2003, 26, 332-42) or gas permeable petri dish as previously described(G-REX flask). In some embodiments, the second amplification (including amplification known as rapid amplification) is performed in T-175 flasks, and may be suspended in about 1X 10 of 150mL of media⁶TIL was added to each T-175 flask. TIL can be cultured in CM and AFM-V media at 1: 1 mixture supplemented with 3000IU/mL IL-2 and 30ng/mL anti-CD 3. The T-175 flask can be heated at 37 ℃ with 5% CO₂And (4) carrying out incubation. On day 5, half of the medium was replaced with 50/50 medium containing 3000IU/mL IL-2. In some embodiments, on day 7, cells from two T-175 flasks may be combined in a 3L bag and 300mL AIM V containing 5% human AB serum and 3000IU/mL IL-2 added to 300mL TIL suspension. The number of cells in each bag was counted daily or every 2 days, and fresh medium was added to maintain the cell count at 0.5X 10⁶To 2.0X 10⁶Individual cells/mL.

In one embodiment, the second amplification (which may include amplification referred to as REP, and those mentioned in step D of FIG. 7) may be performed at a capacity of 500mL, 100cm on a gas permeable silicon substrate ²Gas permeable flask of (G-Rex 100, commercially available from WilsonWolf Manufacturing Corporation, New Bladeton, MN, USA), 5X 10⁶Or 10X 10⁶TIL can be cultured with PBMC in 400mL of 50/50 medium supplemented with 5% human AB serum, 3000IU/mL IL-2 and 30ng/mL anti-CD 3(OKT 3). The G-REX 100 flask can be used for 5% CO at 37 DEG C₂And (4) carrying out incubation. On day 5, 250mL of supernatant can be removed and placed in a centrifuge bottle and centrifuged at 1500rpm (491 Xg) for 10 minutes. The TIL pellet can be resuspended in 150mL fresh medium containing 5% human AB serum, 3000IU/mL IL-2, and added back to the original G-REX 100 flask. When TIL is continuously expanded in G-REX 100 flasks, on day 7, TIL in each G-REX 100 can be suspended in 300mL of medium present in the respective flask, and the cell suspension can be divided into 3 100mL aliquots that can be used to inoculate 3G-REX 100 flasks. Then 150mL AIM-V containing 5% human AB serum and 3000IU/mL IL-2 can be added to each flask. The G-Rex 100 flask can be used for 5% CO at 37 DEG C₂After 4 days, 150mL of AIM-V containing 3000IU/mL IL-2 can be added to each G-REX 100 flask. Can be atCells were harvested on day 14 of culture.

In one embodiment, the second amplification (including amplification referred to as REP) is performed in a flask; wherein a large amount of TIL is mixed with a 100-or 200-fold excess of inactivated feeder cells, 30mg/mL OKT3 anti-CD 3 antibody and 3000IU/mL IL-2 in 150mL of culture medium. In some embodiments, the media change is performed until the cells are transferred to an alternate growth chamber. In some embodiments, fresh medium respiration to replace 2/3 of the medium. In some embodiments, as discussed more fully below, the alternative growth chamber includes a G-REX flask and a gas permeable container.

In one embodiment, a second amplification (including amplification referred to as REP) is performed, and further comprising the step of selecting for TILs with superior tumor reactivity. Any selection method known in the art may be used. For example, the methods described in U.S. patent application publication No. 2016/0010058a1 (the disclosure of which is incorporated herein by reference) can be used to select TILs with excellent tumor reactivity.

Alternatively, cell viability assays can be performed after a second amplification (including amplification referred to as REP amplification) using standard assays known in the art. For example, a trypan blue exclusion assay (trypan blue exclusion assay) can be performed on a large number of TIL samples, which selectively label dead cells and allow viability assessment. In some embodiments, TIL samples can be counted and viability detected using a Cellometer K2 automated cell counter (Nexcelom Bioscience, Lawrence, MA). In some embodiments, viability is determined according to the Cellometer K2 ImageCytometer Automatic Cell Counter (Cellometer K2 image cytometer Automatic Cell Counter) protocol described, for example, in example 15.

In some embodiments, a second amplification of TIL, including amplification referred to as REP, can be performed using a T-175 flask and vented bag as previously described (Tran KQ, Zhou J, Durflinger KH et al, 2008, J.Immunother., 31: 742-751 and Dudley ME, Wunderlich JR, Shelton TE et al, 2003, J.Immunother., 26: 332-342) or a gas permeable G-REX flask. In some embodiments, the second amplification is performed using a flask. In some embodiments, the second amplification isThis was done using a gas permeable G-REX flask. In some embodiments, the second amplification is performed in a T-175 flask, which will be about 1X 10⁶TIL was suspended in about 150mL of medium and added to each T-175 flask. TIL was mixed with irradiated (50Gy) allogeneic PBMC (as "feeder" cells) at 1: 100 ratio, cells were cultured in CM and AIM-V medium at 1: 1 mixture (50/50 medium) supplemented with 3000IU/mL IL-2 and 30ng/mL anti-CD 3. A T-175 flask was kept at 37 ℃ with 5% CO₂And (4) carrying out incubation. In some embodiments, on day 5, half of the medium is replaced with 50/50 medium containing 3000IU/mL IL-2. In some embodiments, on day 7, cells from 2T-175 flasks were combined in a 3L bag and 300mL AIM-V containing 5% human AB serum and 3000IU/mL IL-2 was added to 300mL TIL suspension. The number of cells in each bag can be counted daily or every 2 days, and fresh medium can be added to maintain the cell count at about 0.5X 10 ⁶To about 2.0X 10⁶Individual cells/mL.

In some embodiments, the second amplification (including amplification referred to as REP) is at least 100cm²In a 500 mL-volume flask (G-REX 100, Wilson Wolf) with a gas-permeable silicon bottom (FIG. 1), about 5X 10⁶Or 10X 10⁶TIL and irradiated allogeneic PBMC were mixed at 1: 100 were cultured in 400mL of 50/50 medium supplemented with 3000IU/mL IL-2 and 30ng/mL anti-CD 3. G-REX100 flask at 37 deg.C, 5% CO₂And (4) carrying out incubation. In some embodiments, on day 5, 250mL of supernatant is removed and placed in a centrifuge bottle and centrifuged at 1500rpm (491g) for 10 minutes. The TIL pellet can then be resuspended in 150mL of fresh 50/50 medium containing 3000IU/mL IL-2 and added back to the original G-REX100 flask. In the embodiment where TIL is continuously expanded in G-REX100 flasks, on day 7, TIL in each G-REX100 is suspended in 300mL of medium present in the respective flask and the cell suspension is divided into 3 100mL aliquots used to inoculate 3G-REX 100 flasks. Then to each flask was added 150mL AIM-V containing 5% human AB serum and 3000IU/mL IL-2. G-REX100 flask at 37 deg.C, 5% CO₂After 4 days, the flask containing G-REX100 was added to each flask 150mL AIM-V with 3000IU/mL IL-2. Cells were harvested on day 14 of culture.

Multiple antigen receptors for T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene fragments: v (variable region), D (variable region), J (connecting region) and C (constant region) determine the binding specificity and downstream applications of immunoglobulins and T Cell Receptors (TCRs). The present invention provides methods of producing TILs that demonstrate and increase the diversity of T cell banks. In some embodiments, the TILs obtained by the present methods exhibit increased diversity in the T cell repertoire. In some embodiments, the TIL obtained from the second expansion exhibits an increase in diversity of the T cell pool. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, the immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, the diversity is T cell receptor diversity. In some embodiments, the diversity is a diversity of T cell receptors selected from one of alpha, beta, gamma, and receptor. In some embodiments, expression of T Cell Receptors (TCR) alpha and/or beta is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, expression of TCRab (i.e., TCR α/β) is increased.

In some embodiments, the second expansion medium (e.g., sometimes referred to as CM2 or second cell culture medium) comprises IL-2, OKT-3, and antigen presenting feeder cells (APCs), as discussed in more detail below.

In some embodiments, the second amplification (e.g., according to step D of fig. 8) is performed in a closed system bioreactor. In some embodiments, the TIL amplification is performed using a closed system, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, for example, the single bioreactor employed is G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, during the second amplification (e.g., according to step D of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu. Msd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium. In some embodiments, during the second amplification (e.g., according to step D of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, during the second amplification (e.g., according to step D of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) may be added to a cell culture medium comprising TIL and other agents in an amount selected from the group consisting of: mu.M sd-RNA/10,000TIL, 0.5. mu.M sd-RNA/10,000TIL, 0.75. mu.M sd-RNA/10,000TIL, 1. mu.M sd-RNA/10,000TIL, 1.25. mu.M sd-RNA/10,000TIL, 1.5. mu.M sd-RNA/10,000TIL, 2. mu.M sd-RNA/10,000TIL, 5. mu.M sd-RNA/10,000TIL, or 10. mu.M sd-RNA/10,000 TIL. In some embodiments, during the second amplification (e.g., according to step D of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

1. Feeder cells and antigen presenting cells

In one embodiment, the second expansion step described herein (e.g., including those described in step D of fig. 8, for example, as well as those referred to as REP) requires an excess of feeder cells during the REP TIL expansion and/or during the second expansion. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) obtained from standard whole blood units of healthy donors. PBMCs are obtained using standard methods (e.g., Ficoll-Paque gradient separation).

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used for the REP step, which provides an exemplary protocol for assessing replication insufficiency (replication incompetence) of irradiated allogeneic PBMCs, as described in the examples.

In some embodiments, if the total number of viable cells at day 14 is less than the initial number of viable cells put into culture at day 0 of the REP and/or day 0 of the second expansion (i.e., the starting day of the second expansion), the PBMC is considered to be incompetent to replicate and receive PBMCs for the TIL expansion step described herein.

In some embodiments, if the total number of viable cells cultured in the presence of OKT3 and IL-2 on days 7 and 14 is not increased compared to the initial number of viable cells placed in culture on day 0 of REP and/or day 0 of the second expansion (i.e., the start day of the second expansion), the PBMC are considered to be incompetent to replicate and receive PBMCs for the TIL expansion step described herein. In some embodiments, the PBMCs are cultured in the presence of 5ng/mL to 60ng/mL OKT3 antibody and 1000IU/mL to 6000IU/mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 10ng/mL to 50ng/mL OKT3 antibody and 2000IU/mL to 5000IU/mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 20ng/mL to 40ng/mL OKT3 antibody and 2000IU/mL to 4000IU/mL IL-2. In some embodiments, the PBMCs are cultured in the presence of 25ng/mL to 35ng/mLOKT3 antibody and 2500IU/mL to 3500IU/mL IL-2.

In some embodiments, the antigen presenting feeder cells are PBMCs. In some embodiments, the antigen presenting feeder cells are artificial antigen presenting feeder cells. In one embodiment, the ratio of TIL to antigen presenting feeder cells in the second expansion is about 1: 25. about 1: 50. about 1: 100. about 1: 125. about 1: 150. about 1: 175. about 1: 200. about 1: 225. about 1: 250. about 1: 275. about 1: 300. about 1: 325. about 1: 350. about 1: 375. about 1: 400, or about 1: 500. in one embodiment, the ratio of TIL to antigen presenting feeder cells in the second expansion is 1: 50 to 1: 300. in one embodiment, the ratio of TIL to antigen presenting feeder cells in the second expansion is 1: 100 to 1: 200.

In one embodiment, the second amplification procedure described herein requires about 2.5X 10⁹Feeder cell ratio of about 100X 10⁶The ratio of TILs. In another embodiment, the second amplification procedure described herein requires about 2.5X 10⁹Feeder cell ratio of about 50X 10⁶The ratio of TILs. In yet another embodiment, the second amplification procedure described herein requires about 2.5X 10⁹Feeder cells to about 25X 10⁶The ratio of TILs.

In one embodiment, the second expansion step described herein requires an excess of feeder cells during the second expansion. In many embodiments, the feeder cells are Peripheral Blood Mononuclear Cells (PBMCs) obtained from standard whole blood units of healthy donors. PBMCs are obtained using standard methods such as Ficoll-Paque gradient separation. In one embodiment, artificial antigen presenting (aAPC) cells are used instead of PBMCs.

Typically, allogeneic PBMCs are inactivated by irradiation or heat treatment and used in the TIL amplification steps described herein, including, for example, the exemplary steps described in fig. 4, 5, 6, and 7.

In one embodiment, artificial antigen presenting cells are used in place of PBMCs or in combination with PBMCs in the second expansion.

2. Cytokine

As known in the art, the amplification methods described herein typically use media with high doses of cytokines (especially IL-2).

E. Step E: harvesting of TIL

After the second expansion step, the cells may be harvested. In some embodiments, for example, the TIL is harvested after one, two, three, 4, or more amplification steps as provided in fig. 8. In some embodiments, for example, the TIL is harvested after two amplification steps as provided in fig. 8.

TIL may be harvested in any suitable and sterile manner, including, for example, by centrifugation. Methods of harvesting TIL are well known in the art, and any such known method may be used with the methods of the present invention. In some embodiments, the TIL is harvested using an automated system.

Cell collectors and/or cell processing systems are commercially available from a variety of sources, including, for example, Fresenius Kabi, Tomtec Life Science, Perkin Elmer, and Inotech Biosystems International, inc. Any cell-based collector can be used in the methods of the invention. In some embodiments, the cell collector and/or cell processing system is a membrane-based cell collector. In some embodiments, the cells are harvested by a cell processing system, such as the LOVO system (manufactured by Fresenius Kabi). The term "LOVO cell processing system" also refers to any apparatus or device manufactured by any supplier that can pump a solution containing cells through a membrane or filter (e.g., a rotating membrane or rotating filter) in a sterile and/or closed system environment, allowing for continuous flow and cell processing to remove supernatant or cell culture media that is free of sediment. In some embodiments, the cell collector and/or cell processing system can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

In some embodiments, harvesting (e.g., according to step E of fig. 8) is performed in a closed system bioreactor. In some embodiments, the TIL amplification is performed using a closed system, as described herein. In some embodiments, a single bioreactor is employed. In some embodiments, for example, the single bioreactor employed is G-REX-10 or G-REX-100. In some embodiments, the closed system bioreactor is a single bioreactor.

In some embodiments, step E of fig. 8 is performed according to the process described in example 16. In some embodiments, to maintain sterility and closure of the system, the closed system is accessed through a syringe under sterile conditions. In some embodiments, a closed system as described in example 16 is employed.

In some embodiments, the TIL is harvested according to the method described in example 16. In some embodiments, TILs are harvested from day 1 to day 11 using the methods described in section 8.5 (referred to as day 11 TIL harvest in example 16). In some embodiments, TILs are harvested from day 12 to day 22 using the methods described in section 8.12 (referred to as day 22 TIL harvest in example 16).

F. Step F: final formulation/transfer to infusion bag

After steps a through E, provided in the exemplary sequence in fig. 7, and upon completion of the summary as described above and detailed herein, the cells are transferred to a container for administration to a patient. In some embodiments, once a therapeutically sufficient amount of TILs are obtained using the amplification methods described above, they are transferred to a container for administration to a patient.

In one embodiment, the APC-expanded TIL of the present disclosure is used as a pharmaceutical composition for administration to a patient. In one embodiment, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TILs amplified using PBMCs of the present disclosure may be administered by any suitable route known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic.

1. Pharmaceutical compositions, dosages and administration regimens

In one embodiment, the TIL amplified using the methods of the present disclosure is administered to a patient as a pharmaceutical composition. In one embodiment, the pharmaceutical composition is a suspension of TIL in a sterile buffer. TILs amplified using PBMCs of the present disclosure may be administered by any suitable route known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, preferably for about 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal and intralymphatic administration.

Any suitable dose of TIL may be administered. In some embodiments, particularly when the cancer is melanoma, about 2.3 x 10 is administered¹⁰To about 13.7X 10¹⁰TIL population, average about 7.8X 10¹⁰And (3) TIL group. In one embodiment, about 1.2X 10 is administered¹⁰To about 4.3X 10¹⁰And (3) TIL group. In some embodiments, about 3 x 10 is administered¹⁰To about 12X 10¹⁰And (3) TIL group. In some embodiments, about 4 x 10 is administered¹⁰To about 10X 10¹⁰And (3) TIL group. In some embodiments, about 5 x 10 is administered¹⁰To about 8X 10¹⁰And (3) TIL group. In some embodiments, about 6 x 10 is administered¹⁰To about 8X 10¹⁰And (3) TIL group. In some embodiments, about 7 x 10 is administered ¹⁰To about 8X 10¹⁰And (3) TIL group. In some embodiments, the therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰. In some embodiments, particularly when the cancer is melanoma, the therapeutically effective dose is about 7.8 x 10¹⁰And (3) TIL group. In some embodiments, the therapeutically effective dose is about 1.2 x 10¹⁰To about 4.3X 10¹⁰And (3) TIL group. In some embodiments, the therapeutically effective dose is about 3 x 10¹⁰To about 12X 10¹⁰And (3) TIL group. In some embodiments, the therapeutically effective dose is about 4 x 10¹⁰To about 10X 10¹⁰And (3) TIL group. In some embodiments, the therapeutically effective dose is about 5 x 10¹⁰To about 8X 10¹⁰And (3) TIL group. In some embodiments, the therapeutically effective dose is about 6 x 10¹⁰To about 8X 10¹⁰And (3) TIL group. In some embodiments, the therapeutically effective dose is about 7 x 10¹⁰To about 8X 10¹⁰And (3) TIL group.

In some embodiments, the TIL is provided in the pharmaceutical compositions of the invention in an amount of about 1 × 10⁶、2×10⁶、3×10⁶、4×10⁶、5×10⁶、6×10⁶、7×10⁶、8×10⁶、9×10⁶、1×10⁷、2×10⁷、3×10⁷、4×10⁷、5×10⁷、6×10⁷、7×10⁷、8×10⁷、9×10⁷、1×10⁸、2×10⁸、3×10⁸、4×10⁸、5×10⁸、6×10⁸、7×10⁸、8×10⁸、9×10⁸、1×10⁹、2×10⁹、3×10⁹、4×10⁹、5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、1×10¹¹、2×10¹¹、3×10¹¹、4×10¹¹、5×10¹¹、6×10¹¹、7×10¹¹、8×10¹¹、9×10¹¹、1×10¹²、2×10¹²、3×10¹²、4×10¹²、5×10¹²、6×10¹²、7×10¹²、8×10¹²、9×10¹²、1×10¹³、2×10¹³、3×10¹³、4×10¹³、5×10¹³、6×10¹³、7×10¹³、8×10¹³And 9X 10¹³. At one endIn one embodiment, the amount of TIL provided in a pharmaceutical composition of the invention ranges from 1X 10⁶To 5X 10⁶、5×10⁶To 1X 10⁷、1×10⁷To 5X 10⁷、5×10⁷To 1X 10⁸、1×10⁸To 5X 10⁸、5×10⁸To 1X 10⁹、1×10⁹To 5X 10⁹、5×10⁹To 1X 10¹⁰、1×10¹⁰To 5X 10¹⁰、5×10¹⁰To 1X 10¹¹、5×10¹¹To 1X 10¹²、1×10¹²To 5X 10¹²And 5X 10¹²To 1X 10¹³。

In some embodiments, the TIL is provided in a pharmaceutical composition of the invention at a concentration less than, e.g., 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.3%, 0.0002%, or 0.0001%, v/w% or w/v/w of the pharmaceutical composition.

In some embodiments, the concentration of TIL provided in a pharmaceutical composition of the invention is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25%, 19%, 18.75%, 18.50%, 18.25%, 18%, 17.75%, 17.50%, 17.25%, 17%, 16.75%, 16.50%, 16.25%, 16%, 15.75%, 15.50%, 15.25%, 15%, 14.75%, 14.50%, 14%, 13.75%, 13.50%, 13.25%, 12.75%, 12.50%, 12.25%, 12%, 11.75%, 11.50%, 11.25%, 11%, 10.75%, 10.50%, 10.25%, 10%, 9.75%, 9.50%, 9.25%, 9%, 8.75%, 8.50%, 8.25%, 8%, 7.75%, 7.50%, 7.25%, 6.25%, 6.5%, 6.25%, 3.5%, 4.75%, 3.5%, 3.75%, 3.25%, 4.75%, 3.25%, 4.25%, 3.75%, 3.25%, 4.25%, 3.75%, 4.25%, 3.25%, 4% of the pharmaceutical, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w/w, w/v or v/v.

In some embodiments, the TIL is provided in a pharmaceutical composition of the invention at a concentration ranging from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, from about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 21%, from about 0.2% to about 20%, from about 0.3% to about 19%, from about 0.4% to about 18%, from about 0.5% to about 17%, from about 0.6% to about 16%, from about 0.7% to about 15%, from about 0.8% to about 14%, from about 0.9% to about 12%, or from about 1% to about 10% w/v/w of the pharmaceutical composition.

In some embodiments, the TIL is provided in a pharmaceutical composition of the invention at a concentration ranging from about 0.001% to about 10%, from about 0.01% to about 5%, from about 0.02% to about 4.5%, from about 0.03% to about 4%, from about 0.04% to about 3.5%, from about 0.05% to about 3%, from about 0.06% to about 2.5%, from about 0.07% to about 2%, from about 0.08% to about 1.5%, from about 0.09% to about 1%, from about 0.1% to about 0.9% w/w, w/v, or v/v of the pharmaceutical composition.

In some embodiments, TIL is provided in the pharmaceutical compositions of the invention in an amount equal to or less than 10g, 9.5g, 9.0g, 8.5g, 8.0g, 7.5g, 7.0g, 6.5g, 6.0g, 5.5g, 5.0g, 4.5g, 4.0g, 3.5g, 3.0g, 2.5g, 2.0g, 1.5g, 1.0g, 0.95g, 0.9g, 0.85g, 0.8g, 0.75g, 0.7g, 0.65g, 0.6g, 0.55g, 0.5g, 0.45g, 0.4g, 0.35g, 0.3g, 0.25g, 0.2g, 0.15g, 0.1g, 0.09g, 0.08g, 0.008g, 0.04g, 0000.01 g, 0.04g, 0.06g, 0.04g, 0.01g, 0.04g, 0.01g, 0.009g, 0.0.0.0.0.01 g, 0.0.0.01 g, 0.6g, 0.3g, 0.06g, 0.3.

In some embodiments, TIL is provided in a pharmaceutical composition of the invention in an amount greater than 0.0001g, 0.0002g, 0.0003g, 0.0004g, 0.0005g, 0.0006g, 0.0007g, 0.0008g, 0.0009g, 0.001g, 0.0015g, 0.002g, 0.0025g, 0.003g, 0.0035g, 0.004g, 0.0045g, 0.005g, 0.0055g, 0.006g, 0.0065g, 0.007g, 0.0075g, 0.008g, 0.0085g, 0.009g, 0.95g, 0.01g, 0.015g, 0.02g, 0.025g, 0.03g, 0.035g, 0.04g, 0.045g, 0.05g, 060.06 g, 0.005g, 0.01g, 0.015g, 0.15g, 0.085g, 0.15g, 0.7g, 0.06g, 0.7g, 0.5g, 0.15g, 0.7g, 0.15g, 0.7g, 0.15g, 0.7g, 0.15g, 0.7, 8g, 8.5g, 9g, 9.5g or 10 g.

The TIL provided in the pharmaceutical composition of the present invention is effective over a wide dosage range. The precise dosage will depend upon the route of administration, the form of the compound administered, the sex and age of the subject to be treated, the weight of the subject to be treated, and the preferences and experience of the attending physician. Clinically determined doses of TIL may also be used, if appropriate. The amount of the pharmaceutical composition (e.g., the dose of TIL) administered using the methods herein will depend on the human or mammal being treated, the severity of the condition or disorder, the rate of administration, the configuration of the active pharmaceutical ingredient, and the discretion of the prescribing physician.

In some embodiments, the TIL may be administered in a single dose. Such administration may be by injection, for example, intravenous injection. In some embodiments, the TIL may be administered in multiple doses. The dose may be once, twice, three times, four times, five times, six times or more than six times per year. The dose may be monthly, biweekly, weekly, or every 2 days. Administration of TIL may continue as long as necessary.

In some embodimentsAn effective dose of TIL is about 1X 10⁶、2×10⁶、3×10⁶、4×10⁶、5×10⁶、6×10⁶、7×10⁶、8×10⁶、9×10⁶、1×10⁷、2×10⁷、3×10⁷、4×10⁷、5×10⁷、6×10⁷、7×10⁷、8×10⁷、9×10⁷、1×10⁸、2×10⁸、3×10⁸、4×10⁸、5×10⁸、6×10⁸、7×10⁸、8×10⁸、9×10⁸、1×10⁹、2×10⁹、3×10⁹、4×10⁹、5×10⁹、6×10⁹、7×10⁹、8×10⁹、9×10⁹、1×10¹⁰、2×10¹⁰、3×10¹⁰、4×10¹⁰、5×10¹⁰、6×10¹⁰、7×10¹⁰、8×10¹⁰、9×10¹⁰、1×10¹¹、2×10¹¹、3×10¹¹、4×10¹¹、5×10¹¹、6×10¹¹、7×10¹¹、8×10¹¹、9×10¹¹、1×10¹²、2×10¹²、3×10¹²、4×10¹²、5×10¹²、6×10¹²、7×10¹²、8×10¹²、9×10¹²、1×10¹³、2×10¹³、3×10¹³、4×10¹³、5×10¹³、6×10¹³、7×10¹³、8×10¹³And 9X 10¹³. In some embodiments, an effective dose of TIL is in the range of 1 × 10⁶To 5X 10⁶、5×10⁶To 1X 10⁷、1×10⁷To 5X 10⁷、5×10⁷To 1X 10⁸、1×10⁸To 5X 10⁸、5×10⁸To 1X 10⁹、1×10⁹To 5X 10⁹、5×10⁹To 1X 10¹⁰、1×10¹⁰To 5X 10¹⁰、5×10¹⁰To 1X 10¹¹、5×10¹¹To 1X 10¹²、1×10¹²To 5X 10¹²And 5X 10¹²To 1X 10¹³。

In some embodiments, an effective dose of the TIL ranges from about 0.01mg/kg to about 4.3mg/kg, from about 0.15mg/kg to about 3.6mg/kg, from about 0.3mg/kg to about 3.2mg/kg, from about 0.35mg/kg to about 2.85mg/kg, from about 0.15mg/kg to about 2.85mg/kg, from about 0.3mg to about 2.15mg/kg, from about 0.45mg/kg to about 1.7mg/kg, from about 0.15mg/kg to about 1.3mg/kg, from about 0.3mg/kg to about 1.15mg/kg, from about 0.45mg/kg to about 1mg/kg, from about 0.55mg/kg to about 0.85mg/kg, from about 0.65mg/kg to about 0.8mg/kg, from about 0.7mg/kg to about 0.75mg/kg, from about 0.7mg/kg to about 2.85mg/kg, from about 0.65mg/kg, About 1.15mg/kg to about 1.7mg/kg, about 1.3mg/kg to about 1.6mg/kg, about 1.35mg/kg to about 1.5mg/kg, about 2.15mg/kg to about 3.6mg/kg, about 2.3mg/kg to about 3.4mg/kg, about 2.4mg/kg to about 3.3mg/kg, about 2.6mg/kg to about 3.15mg/kg, about 2.7mg/kg to about 3mg/kg, about 2.8mg/kg to about 3mg/kg, or about 2.85mg/kg to about 2.95 mg/kg.

In some embodiments, an effective dose of the TIL ranges from about 1mg to about 500mg, about 10mg to about 300mg, about 20mg to about 250mg, about 25mg to about 200mg, about 1mg to about 50mg, about 5mg to about 45mg, about 10mg to about 40mg, about 15mg to about 35mg, about 20mg to about 30mg, about 23mg to about 28mg, about 50mg to about 150mg, about 60mg to about 140mg, about 70mg to about 130mg, about 80mg to about 120mg, about 90mg to about 110mg, about 95mg to about 105mg, about 98mg to about 102mg, about 150mg to about 250mg, about 160mg to about 240mg, about 170mg to about 230mg, about 180mg to about 220mg, about 190mg to about 210mg, about 195mg to about 205mg, or about 198 to about 207 mg.

An effective amount of TIL may be administered in a single or multiple doses by any acceptable mode of administration of agents having similar utility, including intranasal and transdermal routes, by intra-arterial injection, intravenous, intraperitoneal, parenteral, intramuscular, subcutaneous, topical, by transplantation or by inhalation.

G. Optional cell culture Medium Components

1. anti-CD 3 antibodies

In some embodiments, the media used in the amplification methods described herein (including those referred to as REP, see, e.g., panel a) also comprises an anti-CD 3 antibody. Binding of anti-CD 3 antibodies to IL-2 induces T cell activation and cell division in the TIL population. This effect can be seen with full length antibodies as well as Fab and F (ab') 2 fragments, the former being generally preferred. See, e.g., Tsoukas et al, j.immunol.1985, 135, 1719, the entire contents of which are incorporated herein by reference in their entirety.

As will be appreciated by those skilled in the art, there are many suitable anti-human CD3 antibodies that can be used in the present invention, including anti-human CD3 polyclonal and monoclonal antibodies from various mammals, including but not limited to murine, human, primate, rat, and canine antibodies. In a specific embodiment, an OKT3 anti-CD 3 antibody (commercially available from Ortho-McNeil, Raritan, NJ or Miltenyi Biotech, Aubum, Calif.) is used.

2.4-1BB (CD137) agonists

In one embodiment, the TNFRSF agonist is a 4-1BB (CD137) agonist. The 4-1BB agonist may be any 4-1BB binding molecule known in the art. The 4-1BB binding molecule may be a monoclonal antibody or a fusion protein capable of binding to human or mammalian 4-1 BB. The 4-1BB agonist or 4-1BB binding molecule may comprise any isotype of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin heavy chain. A4-1 BB agonist or 4-1BB binding molecule may have both a heavy chain and a light chain. As used herein, the term "binding molecule" also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies or chimeric antibodies and antibody fragments (e.g., Fab fragments, F (ab') fragments, fragments produced from Fab expression libraries, epitope-binding fragments of any of the above), and engineered versions of antibodies (e.g., scFv molecules) that bind to 4-1 BB. In one embodiment, the 4-1BB agonist is an antigen binding protein, which is a fully human antibody. In one embodiment, the 4-1BB agonist is an antigen binding protein, which is a humanized antibody. In some embodiments, the 4-1BB agonist used in the methods and compositions of the present disclosure comprises: anti-4-1 BB antibodies, human anti-4-1 BB antibodies, mouse anti-4-1 BB antibodies, mammalian anti-4-1 BB antibodies, monoclonal anti-4-1 BB antibodies, polyclonal anti-4-1 BB antibodies, chimeric anti-4-1 BB antibodies, anti-4-1 BB fibronectin (Adnectin), anti-4-1 BB domain antibodies, single chain anti-4-1 BB fragments, heavy chain anti-4-1 BB fragments, light chain anti-4-1 BB fragments, anti-4-1 BB fusion proteins, and fragments, derivatives, conjugates, variants or biological analogs thereof. Agonistic anti-4-1 BB antibodies are known to induce a strong immune response. Lee et al, PLOS One 2013,8, e 69677. In a preferred embodiment, the 4-1BB agonist is an agonistic anti-4-1 BB humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line). In one embodiment, the 4-1BB agonist is EU-101(Eutilex co.ltd.), urotuzumab or Uluzumab, or a fragment, derivative, conjugate, variant or biological analog thereof. In a preferred embodiment, the 4-1BB agonist is urotuzumab or Uluzumab, or a fragment, derivative, conjugate, variant or biological analog thereof.

In a preferred embodiment, the 4-1BB agonist or 4-1BB binding molecule may also be a fusion protein. In a preferred embodiment, a multimeric 4-1BB agonist (e.g., a trimeric or hexameric 4-1BB agonist (with three or six ligand binding domains)) can induce superior receptor (4-1BBL) aggregation and internal cell signaling complex formation as compared to an agonistic monoclonal antibody, which typically has two ligand binding domains. Fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins, either trimeric (trivalent) or hexameric (or hexavalent) or larger, are described, for example, in giefers et al, mol.

Agonistic 4-1BB antibodies and fusion proteins are known to induce strong immune responses. In a preferred embodiment, the 4-1BB agonist is a monoclonal antibody or fusion protein that specifically binds to 4-1BB antigen in a manner sufficient to reduce toxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular cytotoxicity (ADCC), e.g., NK cell cytotoxicity. In some embodiments, the 4-1BB agonist is an agonistic 4-1BB monoclonal antibody or fusion protein that abrogates antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that abrogates Complement Dependent Cytotoxicity (CDC). In some embodiments, the 4-1BB agonist is an agonist 4-1BB monoclonal antibody or fusion protein that abrogates the functionality of the Fc region.

In some embodiments, the 4-1BB agonist is characterized by binding to human 4-1BB (SEQ ID NO: 9) with high affinity and agonistic activity. In one embodiment, the 4-1BB agonist is a binding molecule that binds to human 4-1BB (SEQ ID NO: 9). In one embodiment, the 4-1BB agonist is a binding molecule that binds murine 4-1BB (SEQ ID NO: 10). Table 3 summarizes the amino acid sequences of 4-1BB antigens bound to 4-1BB agonists or binding molecules.

Table 3: amino acid sequence of 4-1BB antigen

In some embodiments, the compositions, processes and methods comprise K_DA 4-1BB agonist, K, that binds human or murine 4-1BB at about 100pM or less_DBinding to human or murine 4-1BB, K at about 100pM or less_DK is less than 90pM_DA 4-1BB agonist, K, that binds human or murine 4-1BB at about 80pM or less_DA 4-1BB agonist, K, that binds human or murine 4-1BB at less than about 70pM_DA 4-1BB agonist, K, that binds human or murine 4-1BB at less than about 60pM_DA 4-1BB agonist, K, that binds human or murine 4-1BB at about 50pM or less_DA 4-1BB agonist or K that binds human or murine 4-1BB at less than about 40pM_DA 4-1BB agonist that binds human or murine 4-1BB at about 30pM or less.

In some embodiments, the compositions, processes and methods comprise _assocIs about 7.5X 10⁵4-1BB agonists binding to human or murine 4-1BB, k_assocIs about 7.5X 10⁵4-1BB agonists binding to human or murine 4-1BB, k_assocIs about 8X 10⁵4-1BB agonists binding to human or murine 4-1BB, k_assocIs about 8.5X 10⁵4-1BB agonists binding to human or murine 4-1BB, k_assocIs about 9X 10⁵4-1BB agonists binding to human or murine 4-1BB, k_assocIs about 9.5X 10⁵4-1BB agonists or k binding to human or murine 4-1BB at 1/M.s or higher_assocIs about 1X 10⁶1/M.s or greater 4-1BB agonists that bind human or murine 4-1 BB.

In some embodiments, the compositions, processes and methods comprise_dissocIs about 2X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.1X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.2X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.3X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.4X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.5X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.6X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s _dissocIs about 2.7X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.8X 10^-54-1BB agonists, k, that bind human or murine 4-1BB at less than 1/s_dissocIs about 2.9X 10^-54-1BB agonists or k binding to human or murine 4-1BB at less than 1/s_dissocIs about 3X 10^-54-1BB agonists that bind human or murine 4-1BB at less than 1/s.

In some embodiments, the compositions, processes, and methods comprise IC₅₀4-1BB agonists, IC, that bind human or murine 4-1BB at less than about 10nM₅₀4-1BB agonists, IC, that bind human or murine 4-1BB at less than about 9nM₅₀4-1BB agonists, IC, that bind human or murine 4-1BB at less than about 8nM₅₀4-1BB agonists, IC, that bind human or murine 4-1BB at less than about 7nM₅₀Binding to human or murine 4-1BB at less than about 6nM4-1BB agonists, IC₅₀4-1BB agonists, IC, that bind human or murine 4-1BB at less than about 5nM₅₀4-1BB agonists, IC, that bind human or murine 4-1BB at less than about 4nM₅₀4-1BB agonists, IC, that bind human or murine 4-1BB at less than about 3nM₅₀4-1BB agonists or ICs that bind human or murine 4-1BB at less than about 2nM₅₀4-1BB agonists that bind human or murine 4-1BB are less than about 1 nM.

In a preferred embodiment, the 4-1BB agonist is Utuzumab (also known as PF-05082566 or MOR-7480) or a fragment, derivative, variant, or biological analog thereof. Urotuzumab is commercially available from Pfizer, inc. Utositumumab is immunoglobulin G2-lambda, anti [ homo sapiens TNFRSF9 (tumor necrosis factor receptor (TNFR) superfamily member 9, 4-1BB, T cell antigen ILA, CD137) ]Homo sapiens (fully human) monoclonal antibodies. The amino acid sequence of the urotuzumab is listed in table 4. The urotuzumab comprises: glycosylation sites at Asn59 and Asn 292; at positions 22-96 (V)_H-V_L) 143-199 position (C)_H1-C_L) 256-316 bits (C)_H2) And 362-420 bits (C)_H3) An intrachain disulfide bond of (a); at the 22'-87' position (V)_H-V_L) And 136'-195' (C)_H1-C_L) The light chain intrachain disulfide bond of (a); interchain heavy chain-heavy chain disulfide bonds at positions 218-218, 219-219, 222-222, and 225-225 of the IgG2A isoform, at positions 218-130, 219-219, 222-222, and 225-225 of the IgG2A/B isoform, and at positions 219-130(2), 222-222, and 225-225 of the IgG2B isoform; and interchain heavy-light disulfide bonds at positions 130-213 '(2) of the IgG2A isoform, positions 218-213' and 130-213 'of the IgG2A/B isoform, and positions 218-213' (2) of the IgG2B isoform. The preparation and properties of urotuzumab, variants and fragments thereof are described in U.S. patent nos. 8,821,867, 8,337,850 and 9,468,678; and international patent application publication No. WO2012/032433a1, the respective disclosures of which are incorporated herein by reference. Preclinical characterization of urotuzumab was described by Fisher et al, Cancer Immunolog.&Immunoher.2012, 61, 1721-33. Current clinical trials of urotuzumab in various hematological and solid tumor indications include the u.s.national Institutes of Health clinical trials.gov identifier NCT02444793, c, NCT01307267, NCT02315066 and NCT 02554812.

In one embodiment, the 4-1BB agonist comprises SEQ ID NO: 11 and SEQ ID NO: 12, light chain. In one embodiment, the 4-1BB agonist comprises a peptide having SEQ ID NO: 11 and SEQ ID NO: 12 or an antigen-binding fragment thereof, a Fab fragment, a single chain variable fragment (scFv), a variant thereof, or a conjugate thereof. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 11 and SEQ ID NO: 12 has at least 99% identity to the heavy and light chains. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 11 and SEQ ID NO: 12 has at least 98% identity to the heavy and light chains. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 11 and SEQ ID NO: 12 has at least 97% identity to the heavy and light chains. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 11 and SEQ ID NO: 12 has at least 96% identity to the heavy and light chains. In one embodiment, the 4-1BB agonist comprises a peptide that is identical to SEQ id no: 11 and SEQ ID NO: 12 has at least 95% identity to the heavy and light chains.

In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of urotuzumab. In one embodiment, the 4-1BB agonist heavy chain variable region (V)_H) Comprises the amino acid sequence of SEQ ID NO: 13, 4-1BB agonist light chain variable region (V)_L) Comprises the amino acid sequence of SEQ ID NO: 14, and conservative amino acid substitutions thereof. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 13 and SEQ ID NO: 14 has at least 99% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 13 and SEQ ID NO: 14 has at least 98% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 13 and SEQ ID NO: 14 has at least 97% of the same V_HAnd V_LAnd (4) a zone. In one embodiment the 4-1BB agonist comprises a peptide corresponding to SEQ ID NO: 13 and SEQ ID NO: 14 has at least 96% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 13 and SEQ ID NO: 14 has at least 95% identity with V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises an scFv antibody comprising an amino acid sequence that is identical to SEQ ID NO: 13 and SEQ ID NO: 14 has at least 99% of the same V _HAnd V_LAnd (4) a zone.

In one embodiment, the 4-1BB agonist comprises a peptide having SEQ ID NO: 15. SEQ ID NO: 16 and SEQ ID NO: 17 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 18. SEQ ID NO: 19 and SEQ ID NO: 20 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to Utolumab. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference pharmaceutical product or a reference biological product and comprising one or more post-translational modifications as compared to the reference pharmaceutical product or the reference biological product, wherein the reference pharmaceutical product or the reference biological product is utolimumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or submitted authorized 4-1BB agonist antibody, wherein the 4-1BB agonist antibody is provided in a formulation different from a reference pharmaceutical product or reference biological product formulation, wherein the reference pharmaceutical product or reference biological product is utoluzumab. The 4-1BB agonist antibody may be authorized by the drug regulatory agency (e.g., the U.S. FDA and/or European Union EMA). In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is utolimumab. In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is utolimumab.

Table 4: amino acid sequence of 4-1BB agonist antibody related to urotuzumab

In a preferred embodiment, the 4-1BB agonist is the monoclonal antibody Uluzumab (also known as BMS-663513 and 20H4.9.h4a) or a fragment, derivative, variant, or biological analog thereof. Uribritumumab is commercially available from Bristol-Myers Squibb, Inc. and Creative Biolabs, Inc. Urru monoclonal antibody is immunoglobulin G4-kappa, anti [ homo sapiens TNFRSF9 (tumor necrosis factor receptor superfamily member 9, 4-1BB, T cell antigen ILA, CD137)]Homo sapiens (fully human) monoclonal antibodies. The amino acid sequence of the Uruguzumab is listed in Table 5. The Urru monoclonal antibody comprises: an N-glycosylation site at positions 298 (and 298'); at positions 22-95 (V)_H-V_L) 148-_H1-C_L) 262 and 322 (C)_H2) And 368-_H3) (and 22 "-95" positions, 148 "-204" positions, 262 "-322" positions, and 368 "-426" positions); at the 23'-88' position (V)_H-V_L) And 136'-196' (C)_H1-C_L) (and disulfide bridges in the light chain at positions 23 '"-88'" and 136 '"-196'"; the interchain heavy-chain disulfide bond at position 227-; and interchain heavy-light disulfide bonds at positions 135-216 'and 135' -216 ". The preparation and properties of Uruguzumab, and variants and fragments thereof, are described in U.S. Pat. Nos. 7,288,638 and 8,962,804, the disclosures of which are incorporated herein by reference. Preclinical and clinical features of Uriluzumab are described in Segal et al, Clin 6, available from http: // dx.doi.org/10.1158/1078-0432. CCR-16-1272. Current clinical trials of ureuzumab in various hematological and solid tumor indications include u.s.national institutes of Health clinical rials.gov identifier NCT01775631, NCT02110082, NCT02253992, and NCT 01471210.

In one embodiment, the 4-1BB agonist comprises SEQ ID NO: 21 and SEQ ID NO: 22, light chain as given. In one embodiment, the 4-1BB agonist comprises a peptide having SEQ ID NO: 21 and SEQ ID NO: 22 or an antigen-binding fragment thereof, a Fab fragment, a single chain variable fragment (scFv), a heavy chain and a light chain of a variant or a conjugate. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 21 and SEQ ID NO: 22 has at least 99% identity to the light and heavy chains. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 21 and SEQ ID NO: 22 has at least 98% identity to the light and heavy chains. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 21 and SEQ ID NO: 22 has at least 97% identity to the light and heavy chains. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 21 and SEQ ID NO: 22 has at least 96% identity to the light and heavy chains. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 21 and SEQ ID NO: 22 has at least 95% identity to the heavy and light chains.

In one embodiment, the 4-1BB agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of udeuzumab. In one embodiment, the 4-1BB agonist heavy chain variable region (V)_H) Comprises the amino acid sequence of SEQ ID NO: 23, 4-1BB agonist light chain variable region (V)_L) Comprises the amino acid sequence of SEQ ID NO: 24, and conservative amino acid substitutions thereof. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 23 and SEQ ID NO: 24 has at least 99% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 23 and SEQ ID NO: 24 has at least 98% of the same V_HAnd V_LAnd (4) a zone. In a fruitIn embodiments, the 4-1BB agonist comprises a peptide sequence substantially identical to SEQ ID NO: 23 and SEQ ID NO: 24 has at least 97% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 23 and SEQ ID NO: 24 has at least 96% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises a sequence that is identical to SEQ ID NO: 23 and SEQ ID NO: 24 has at least 95% identical V_HAnd V_LAnd (4) a zone. In one embodiment, the 4-1BB agonist comprises a scFv antibody that binds to the amino acid sequences of SEQ ID NOs: 23 and SEQ ID NO: 24 has at least 99% of the same V _HAnd V_LAnd (4) a zone.

In one embodiment, the 4-1BB agonist comprises a peptide having SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 28, SEQ ID NO: 29 and SEQ ID NO: 30 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to Uluzumab. In one embodiment, the biosimilar monoclonal antibody comprises a 4-1BB antibody, the 4-1BB antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to a reference pharmaceutical product or a reference biological product and comprising one or more post-translational modifications as compared to the reference pharmaceutical product or the reference biological product, wherein the reference pharmaceutical product or the reference biological product is ulivolumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or submitted authorized 4-1BB agonist antibody, wherein the 4-1BB agonist antibody formulation is different from a reference pharmaceutical product or reference biological product formulation, wherein the reference pharmaceutical product or reference biological product is ulirubizumab. The 4-1BB agonist antibody may be authorized by the drug regulatory agency (e.g., the U.S. FDA and/or European Union EMA). In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is Uluzumab. In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is Uluzumab.

Table 5: amino acid sequence of 4-1BB agonist antibody related to Urru monoclonal antibody

In one embodiment, the 4-1BB agonist is selected from: 1D8, 3Elor, 4B4(BioLegend 309809), H4-1BB-M127(BD Pharmingen 552532), BBK2(Thermo Fisher MS621PABX), 145501(Leinco technologies B591), the antibody produced by the cell line deposited under ATCC number HB-11248 and disclosed in U.S. Pat. No. 145501, 5F 145501 (BioLegend 145501), C145501-485 (BD Pharmingen 145501), the antibody disclosed in U.S. patent application publication No. US 2005/145501, the antibody disclosed in U.S. Pat. No. 145501 (e.g. 20H4.9-IgG 145501 (BMS-145501)), the antibody disclosed in U.S. Pat. No. 145501 (e.g. 4E 145501 or BMS-145501), the antibody disclosed in U.S. Pat. No. 145501, the antibody disclosed in U.S. Pat. No. 4E 145501 (e.S. BMS. 4E 145501) or the antibody disclosed in BMS-145501), the antibody disclosed in U.S. Pat. 3H 3-145501, or the antibody disclosed in U.S. Pat. 3E 145501, such as 145501, or the antibody disclosed in U.S. 3-145501, such, Antibodies disclosed in U.S. patent No. 6,974,863 (e.g., 53a 2); antibodies disclosed in U.S. patent 6,210,669 (e.g., 1D8, 3B8, or 3E1), antibodies described in U.S. patent 5,928,893, antibodies disclosed in U.S. patent 6,303,121, antibodies disclosed in U.S. patent 6,569,997, antibodies disclosed in international patent application publication nos. WO2012/177788, WO2015/119923, and WO2010/042433, and fragments, derivatives, conjugates, variants, or biological analogs thereof, wherein the respective disclosures in the foregoing patents or patent application publications are incorporated herein by reference in their entirety.

In one embodiment, the 4-1BB agonist is described in international patent application publication nos. WO2008/025516a1, WO2009/007120a1, WO2010/003766a1, WO2010/010051a1, and WO2010/078966a 1; U.S. patent application publication nos. US2011/0027218a1, US2015/0126709a1, US2011/0111494a1, US2015/0110734a1, and US2015/0126710a 1; and 4-1BB agonistic fusion proteins of U.S. Pat. Nos. 9,359,420, 9,340,599, 8,921,519, and 8,450,460, the disclosures of which are incorporated herein by reference.

In one embodiment, the 4-1BB agonist is a 4-1BB agonistic fusion protein as described by structure I-A (C-terminal Fc antibody fragment fusion protein) or structure I-B (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biological analog thereof:

in structures I-A and I-B, the cylinder refers to a single polypeptide binding domain. Structures I-A and I-B comprise three linearly linked TNFRSF binding domains derived, for example, from 4-1BBL or antibodies that bind to 4-1BB, which fold to form a trivalent protein, which is then passed through IgG1-Fc (including C)_H3 and C _H2 domain) is linked to a second trivalent protein and then used to link the two trivalent proteins together by disulfide bonds (small elongated ovals), thereby stabilizing the structure and providing an agonist capable of binding the intracellular signaling domains of the six receptors and the signaling protein together to form a signaling complex. The TNFRSF binding domain (shown as a cylinder) can be an scFv domain comprising, for example, V joined by a linker _HAnd V_LA chain, the linker may comprise hydrophilic residues and Gly and Ser sequences for flexibility and Glu and Lys for solubility. Any scFv domain design can be used, for example as described in de Marco, microbiological Cell industries, 2011, 10, 44; ahmad et al, Clin.&Dev.immunol.2012,980250; monnier et al, Antibodies, 2013, 2, 193- "208; as well as those incorporated by reference elsewhere herein. Fusion protein structures of this form are described in U.S. patent nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference.

The amino acid sequences of the other polypeptide domains of structure I-A are given in Table 6. The Fc domain preferably comprises the entire constant domain (amino acids 17-230 of SEQ ID NO: 31), the entire hinge domain (amino acids 1-16 of SEQ ID NO: 31) or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 31). Preferred linkers for linking C-terminal Fc antibodies may be selected from SEQ ID NO: 32 to SEQ ID NO: 41, comprising a linker suitable for fusion to another polypeptide.

Table 6: amino acid sequence of TNFRSF fusion proteins (including 4-1BB fusion proteins) with C-terminal Fc-antibody fragment fusion protein design (Structure I-A)

The amino acid sequences of the other polypeptide domains of structure I-B are given in Table 7. If the Fc antibody fragment is fused to the N-terminus of a TNRFSF fusion protein (as in structures I-B), the sequence of the Fc module is preferably SED ID NO: 42, and the linker sequence is preferably selected from the group consisting of SED ID NO: 43 to SEQ ID NO: 45 are shown.

Table 7: amino acid sequence of TNFRSF fusion proteins (including 4-1BB fusion proteins) with N-terminal Fc antibody fragment fusion protein design (Structure I-B)

In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain selected from the group consisting of: a variable heavy chain and a variable light chain of urotuzumab, a variable heavy chain and a variable light chain selected from the variable heavy chain and the variable light chain described in table 8, any combination of the aforementioned variable heavy chain and variable chain, and fragments, derivatives, conjugates, variants and biological analogs thereof.

In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain comprising a 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more polypeptides comprising an amino acid sequence according to SEQ ID NO: 46, or a 4-1BB binding domain of the sequence of SEQ ID NO. In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain comprising a soluble 4-1BBL sequence. In one embodiment, a 4-1BB agonist fusion protein according to structures I-A or I-B comprises one or more polypeptides comprising an amino acid sequence according to SEQ ID NO: 47, or a 4-1BB binding domain of the sequence of SEQ ID NO.

In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, the 4-1BB binding domain being a scFv domain comprising FH and VL regions, and the V_HAnd V_LThe regions are respectively identical to SEQ ID NO: 13 and SEQ ID NO: 14, wherein V is at least 95% identical_HAnd V_LThe domains are connected by a linker. In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, wherein the 4-1BB binding domain comprises V_HAnd V_LscFv domain of region V_HAnd V_LThe regions are respectively identical to SEQ ID NO: 23 and SEQ ID NO: 24 is at least 95% identical, wherein V_HAnd V_LThe domains are connected by a linker. In one embodiment, a 4-1BB agonist fusion protein according to structure I-A or I-B comprises more than one 4-1BB binding domain, wherein the 4-1BB binding domain comprises V_HAnd V_LscFv domains of regionsThe V is_HAnd V_LThe zones are respectively associated with V given in Table 8_HAnd V_LThe sequences have at least 95% identity, wherein V_HAnd V_LThe domains are connected by a linker.

Table 8: other polypeptide domains useful as 4-1BB binding domains in fusion proteins or as scFv 4-1BB agonist antibodies

In one embodiment, the 4-1BB agonist is a 4-1BB agonistic single chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain, further comprising an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In one embodiment, the 4-1BB agonist is a 4-1BB agonistic single chain fusion polypeptide comprising (i) a first soluble 4-1BB binding domain, (ii) a first peptide linker, (iii) a second soluble 4-1BB binding domain, (iv) a second peptide linker, and (v) a third soluble 4-1BB binding domain further comprising an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain; wherein each soluble 4-1BB domain lacks a stem (talk) region (which facilitates trimerization and provides a distance to the cell membrane, but is not part of the 4-1BB binding domain), the first peptide linker and the second linker independently have a length of 3 to 8 amino acids.

In one embodiment, the 4-1BB agonist is a 4-1BB agonistic single chain fusion polypeptide comprising (i) a first soluble Tumor Necrosis Factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each soluble TNF superfamily cytokine domain lacks a stem region, the first and second peptide linkers independently having a length of 3 to 8 amino acids, wherein each TNF superfamily cytokine domain is a 4-1BB binding domain.

In one embodiment, the 4-1BB agonist is a 4-1BB agonistic scFv antibody comprising a heavy chain variable region with any of the foregoing V_LAny of the foregoing V linked by domains_HA domain.

In one embodiment, the 4-1BB agonist is a 4-1BB agonist antibody to BPS Bioscience catalog number 79097-2, commercially available from BPS Bioscience (San Diego, Calif., USA). In one embodiment, the 4-1BB agonist is a 4-1BB agonist antibody to Creative Biolabs catalog number MOM-18179, commercially available from Creative Biolabs (Shirley, NY, USA).

OX40(CD134) agonists

In one embodiment, the TNFRSF agonist is an OX40(CD134) agonist. The OX40 agonist can be any OX40 binding molecule known in the art. The OX40 binding molecule can be a monoclonal antibody or fusion protein capable of binding to human or mammalian OX 40. The OX40 agonist or OX40 binding molecule can comprise any isotype of immunoglobulin molecule (e.g., IgG1, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass of immunoglobulin heavy chain. An OX40 agonist or OX40 binding molecule may have both heavy and light chains. As used herein, the term "binding molecule" also includes antibodies (including full length antibodies), monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies or chimeric antibodies and antibody fragments (e.g., Fab fragments, F (ab') fragments, fragments produced by a Fab expression library, epitope-binding fragments of any of the above), as well as engineered versions of antibodies (e.g., scFv molecules) that bind to OX 40. In one embodiment, the OX40 agonist is an antigen binding protein that is a fully human antibody. In one embodiment, the OX40 agonist is an antigen binding protein that is a humanized antibody. In some embodiments, the OX40 agonists for use in the methods and compositions of the present disclosure include: anti-OX 40 antibodies, human anti-OX 40 antibodies, mouse anti-OX 40 antibodies, mammalian anti-OX 40 antibodies, monoclonal anti-OX 40 antibodies, polyclonal anti-OX 40 antibodies, chimeric anti-OX 40 antibodies, anti-OX 40 fibronectin, anti-OX 40 domain antibodies, single chain anti-OX 40 fragments, heavy chain anti-OX 40 fragments, light chain anti-OX 40 fragments, anti-OX 40 fusion proteins, and fragments, derivatives, conjugates, variants, or biological analogs thereof. In a preferred embodiment, the OX40 agonist is an agonist anti-OX 40 humanized or fully human monoclonal antibody (i.e., an antibody derived from a single cell line).

In a preferred embodiment, the OX40 agonist or OX40 binding molecule also can be a fusion protein. OX40 fusion proteins comprising an Fc domain fused to OX40L are described, for example, in Sadun et al, j. In a preferred embodiment, multimeric OX40 agonists (e.g., trimeric or hexameric OX40 agonists (with three or six ligand binding domains)) can induce superior receptor (OX40L) clustering and internal cell signaling complex formation compared to agonist monoclonal antibodies (which typically have two ligand binding domains). Fusion proteins comprising three TNFRSF binding domains and IgG1-Fc and optionally further linking two or more of these fusion proteins, either trimeric (trivalent) or hexameric (or hexavalent) or larger, are described, for example, in giefers et al, mol.

Agonist OX40 antibodies and fusion proteins are known to induce strong immune responses. Curti et al, cancer Res.2013, 73, 7189-98. In a preferred embodiment, the OX40 agonist is a monoclonal antibody or fusion protein that specifically binds the OX40 antigen in a manner sufficient to reduce toxicity. In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates antibody-dependent cellular cytotoxicity (ADCC) (e.g., NK cell cytotoxicity). In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates Complement Dependent Cytotoxicity (CDC). In some embodiments, the OX40 agonist is an agonist OX40 monoclonal antibody or fusion protein that abrogates Fc region functionality.

In some embodiments, the OX40 agonist is characterized by binding human OX40(SEQ ID NO: 54) with high affinity and agonist activity. In one embodiment, the OX40 agonist is a binding molecule that binds to human OX40(SEQ ID NO: 54). In one embodiment, the OX40 agonist is a binding molecule that binds to murine OX40(SEQ ID NO: 55). Table 9 summarizes the amino acid sequences of OX40 antigen that binds to OX40 agonists or binding molecules.

Table 9: amino acid sequence of OX40 antigen

In some embodiments, the compositions, processes and methods comprise K_DAn OX40 agonist, K that binds human or murine OX40 at less than about 100pM_DOX40 agonist, K, that binds human or murine OX40 at less than about 90pM_DOX40 agonist, K, that binds human or murine OX40 at less than about 80pM_DOX40 agonist, K, that binds human or murine OX40 at less than about 70pM_DAn OX40 agonist, K that binds human or murine OX40 at less than about 60pM_DOX40 agonist, K, that binds human or murine OX40 at less than about 50pM_DOX40 agonist or K that binds human or murine OX40 at less than about 40pM_DAn OX40 agonist that binds human or murine OX40 at less than about 30 pM.

In some embodiments, the compositions, processes and methods comprise _assocIs about 7.5X 10⁵OX40 agonist binding to human or murine OX40, k at 1/M.s or higher_assocIs about 8X 10⁵OX40 agonist binding to human or murine OX40, k at 1/M.s or higher_assocIs about 8.5X 10⁵OX40 agonist binding to human or murine OX40, k at 1/M.s or higher_assocIs about 9X 10⁵OX40 agonist binding to human or murine OX40, k at 1/M.s or higher_assocIs about 9.5X 10⁵OX40 agonist or k binding to human or murine OX40 at 1/M.s or higher_assocIs about 1X 10⁶1/M·sThe above OX40 agonists that bind human or murine OX 40.

In some embodiments, the compositions, processes and methods comprise_dissocIs about 2X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.1X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.2X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.3X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.4X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.5X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.6X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.7X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s_dissocIs about 2.8X 10^-5OX40 agonist, k, that binds human or murine OX40 at less than 1/s _dissocIs about 2.9X 10^-5OX40 agonist or k binding to human or murine OX40 at less than 1/s_dissocIs about 3X 10^-5OX40 agonists that bind human or murine OX40 at less than 1/s.

In some embodiments, the compositions, processes, and methods comprise IC₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 10nM₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 9nM₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 8nM₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 7nM₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 6nM₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 5nM₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 4nM₅₀OX40 agonist, IC, that binds human or murine OX40 at less than about 3nM₅₀OX40 agonists or ICs that bind human or murine OX40 at less than about 2nM₅₀An OX40 agonist that binds human or murine OX40 at less than about 1 nM.

In some implementationsIn one embodiment, the OX40 agonist is talizelizumab (also known as MEDI0562 or MEDI-0562). Taliximab is commercially available from MedImmune, inc. Tallixizumab is immunoglobulin G1-kappa, anti [ homo sapiens TNFRSF4 (tumor necrosis factor receptor (TNFR) superfamily member 4, OX40, CD134) ]Humanized and chimeric monoclonal antibodies. The amino acid sequence of talizezumab is listed in table 10. The taliximab comprises: complex biantennary CHO-type glycans with fucosylation at the N-glycosylation sites at positions 301 and 301 "; at positions 22-95 (V)_H-V_L) 148-_H1-C_L) 265 sub 325 (C)_H2) And 371-_H3) (and 22 "-95" position, 148 "-204" position, 265 "-325" position and 371 "-429" position); at the 23'-88' position (V)_H-V_L) And positions 134'-194' (C)_H1-C_L) (and positions 23 "'-88"' and 134 "'-194"'); the interchain heavy-chain disulfide bond at the 230-230 'and 233-233' positions; and interchain heavy-light chain disulfide bonds at positions 224-214' and 224' -214' ". Current clinical trials of taliximab in various solid tumor indications include the u.s.national institutes healthcare clinical codes NCT02318394 and NCT 02705482.

In one embodiment, the OX40 agonist comprises a polypeptide consisting of SEQ ID NO: 56 and the heavy chain given by SEQ ID NO: light chain given at 57. In one embodiment, the OX40 agonist comprises a peptide having SEQ ID NOs: 56 and SEQ ID NO: 57 or an antigen-binding fragment thereof, a Fab fragment, a single chain variable fragment (scFv), a heavy chain and a light chain of a variant or conjugate. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 56 and SEQ ID NO: 57 has at least 99% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ id no: 56 and SEQ ID NO: 57 has at least 98% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 56 and SEQ ID NO: 57 has at least 97% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 56 and SEQ ID NO: 57 has at least 96% identity between the heavy and light chains. In one embodiment, the OX40 agonist comprises a peptide that binds to seq id NOs: 56 and SEQ ID NO: 57 has at least 95% identity to the heavy and light chains.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of taliximab. In one embodiment, the OX40 agonist heavy chain variable region (V)_H) Comprises the amino acid sequence of SEQ ID NO: 58 and conservative amino acid substitution thereof, OX40 agonist light chain variable region (V)_L) Comprises the amino acid sequence of SEQ ID NO: 59 and conservative amino acid substitution thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 58 and SEQ ID NO: 59 has at least 99% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ id no: 58 and SEQ ID NO: 59 has at least 98% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 58 and SEQ ID NO: v with at least 97% identity to the sequence shown in 59_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 58 and SEQ ID NO: 59 has at least 96% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 58 and SEQ ID NO: 59 has at least 95% of the same V_HAnd V _LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an scFv antibody comprising an amino acid sequence that differs from SEQ ID NO: 58 and SEQ ID NO: 59 has at least 99% of the same V_HAnd V_LAnd (4) a zone.

In one embodiment, the OX40 agonist comprises: have the amino acid sequences of SEQ ID NO: 60. SEQ ID NO: 61 and SEQ ID NO: 62 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 63. SEQ ID NO: 64 and SEQ ID NO: 65 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof.

In one embodiment, the OX40 agonist is an OXA40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to taliximab. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug product or a reference biological product and comprising one or more post-translational modifications as compared to the reference drug product or the reference biological product, wherein the reference drug product or the reference biological product is talipexizumab. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from a formulation of a reference drug product or reference biological product, wherein the reference drug product or reference biological product is taliximab. OX40 agonist antibodies may be authorized by the drug regulatory agency (e.g., FDA and/or EMA of the european union in the united states). In some embodiments, the biological analog is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is taliximab. In some embodiments, the biological analog is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is taliximab.

Table 10: amino acid sequence of an OX40 agonist antibody related to talizelizumab

In some embodiments, the OX40 agonist is 11D4, which is a fully human antibody commercially available from Pfizer, inc. The preparation and properties of 11D4 are described in U.S. patent nos. 7,960,515, 8,236,930 and 9,028,824, the disclosures of which are incorporated herein by reference. Table 11 lists the amino acid sequence of 11D 4.

In one embodiment, the OX40 agonist comprises a polypeptide consisting of SEQ ID NO: 66 and the heavy chain set forth by SEQ ID NO: 67, light chain. In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence of SEQ ID NO: 66 and SEQ ID NO: 67 or an antigen-binding fragment thereof, a Fab fragment, a single chain variable fragment (scFv), a heavy chain and a light chain of a variant or conjugate. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 66 and SEQ ID NO: 67 has at least 99% sequence identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 66 and SEQ ID NO: 67 has at least 98% sequence identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 66 and SEQ ID NO: 67 has at least 97% sequence identity to the light and heavy chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 66 and SEQ ID NO: 67 has at least 96% sequence identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 66 and SEQ ID NO: 67 has at least 95% sequence identity to the heavy and light chains.

In one embodiment, the OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of 11D 4. In one embodiment, the OX40 agonist heavy chain variable region (V)_H) Comprises the amino acid sequence of SEQ ID NO: 68, OX40 agonist light chain variable region (V)_L) Comprises the amino acid sequence of SEQ ID NO: 69, and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 68 and SEQ ID NO: 69 having at least 99% identity to the sequence shown in_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 68 and SEQ ID NO: 69 is at least 98% identical_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 68 and SEQ ID NO: 69 is shown in sequenceV with at least 97% identity_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 68 and SEQ ID NO: 69 is at least 96 percent identical_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 68 and SEQ ID NO: 69 is at least 95% identical_HAnd V_LAnd (4) a zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence of SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 73, SEQ ID NO: 74 and SEQ ID NO: 75 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency reference 11D 4. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody, the OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug product or a reference biological product and comprising one or more post-translational modifications as compared to the reference drug product or the reference biological product, wherein the reference drug product or the reference biological product is 11D 4. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from a formulation of a reference drug product or reference biological product, wherein the reference drug product or reference biological formulation product is 11D 4. OX40 agonist antibodies may be authorized by the drug regulatory agency (e.g., FDA and/or EMA of the european union in the united states). In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is 11D 4. In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is 11D 4.

Table 11: amino acid sequences of OX40 agonist antibodies related to 11D4

In some embodiments, the OX40 agonist is 18D8, which is a fully human antibody commercially available from Pfizer, inc. The preparation and properties of 18D8 are described in U.S. patent nos. 7,960,515, 8,236,930 and 9,028,824, the disclosures of which are incorporated herein by reference. Table 12 lists the amino acid sequence of 18D 8.

In one embodiment, the OX40 agonist comprises a polypeptide consisting of SEQ ID NO: 76 and the heavy chain set forth by SEQ ID NO: 77, or a light chain. In one embodiment, the OX40 agonist comprises a peptide having SEQ ID NOs: 76 and SEQ ID NO: 77 or an antigen binding fragment thereof, a Fab fragment, a single chain variable fragment (scFv), a heavy chain and a light chain of a variant or a conjugate. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 76 and SEQ ID NO: 77 have at least 99% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises a peptide that binds to seq id NOs: 76 and SEQ ID NO: 77 have at least 98% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 76 and SEQ ID NO: 77 have at least 97% identity to the light and heavy chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 76 and SEQ ID NO: 77 have at least 96% identity to the heavy and light chains. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 76 and SEQ ID NO: 77 have at least 95% identity to the heavy and light chains.

In one embodiment, the OX40 agonist comprises 1The heavy and light chain CDRs or Variable Regions (VRs) of 8D 8. In one embodiment, the OX40 agonist heavy chain variable region (V)_H) Comprises the amino acid sequence of SEQ ID NO: 78, OX40 agonist light chain variable region (V)_L) Comprises the amino acid sequence of SEQ ID NO: 79, and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 78 and SEQ ID NO: 79V having at least 99% identity to the sequence shown_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 78 and SEQ ID NO: 79V with at least 98% identity_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 78 and SEQ ID NO: 79 sequence having at least 97% identity V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 78 and SEQ ID NO: 79 sequence having at least 96% identity V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 78 and SEQ ID NO: 79V having at least 95% identity to the sequence shown_HAnd V_LAnd (4) a zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence of SEQ ID NO: 80. SEQ ID NO: 81 and SEQ ID NO: 82 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 83. SEQ ID NO: 84 and SEQ ID NO: 85 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency reference 18D 8. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody, the OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug product or a reference biological product and comprising one or more post-translational modifications as compared to the reference drug product or the reference biological product, wherein the reference drug product or the reference biological product is 18D 8. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biological analog is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from a formulation of a reference drug product or reference biological product, wherein the reference drug product or reference biological product is 18D 8. OX40 agonist antibodies may be authorized by the drug regulatory agency (e.g., FDA and/or EMA of the european union in the united states). In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is 18D 8. In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is 18D 8.

Table 12: amino acid sequences of OX40 agonist antibodies related to 18D8

In some embodiments, the OX40 agonist is Hu119-122, a humanized antibody commercially available from GlaxoSmithKline plc. The preparation and properties of Hu119-122 are described in U.S. patent nos. 9,006,399 and 9,163,085 and international patent publication No. WO2012/027328, the disclosures of which are incorporated herein by reference. Table 13 lists the amino acid sequences of Hu 119-122.

In one embodiment, an OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of Hu 119-122. In one embodiment, the OX40 agonist heavy chain variable region (V)_H) Comprises the amino acid sequence of SEQ ID NO: 86, OX40 agonist light chain variable region (V)_L) Comprises the amino acid sequence of SEQ ID NO: 87 and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 86 and SEQ ID NO: 87 toV showing at least 99% identity of the sequence_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 86 and SEQ ID NO: 87 has a V of at least 98% identity_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ id no: 86 and SEQ ID NO: 87 has at least 97% identity to the sequence shown in V _HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 86 and SEQ ID NO: 87 has a V of at least 96% identity_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 86 and SEQ ID NO: 87 has at least 95% identity with the sequence V_HAnd V_LAnd (4) a zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence of SEQ ID NO: 88. SEQ ID NO: 89 and SEQ ID NO: 90 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 91. SEQ ID NO: 92 and SEQ ID NO: 93 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to Hu 119-122. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug product or a reference biological product and comprising one or more post-translational modifications as compared to the reference drug product or the reference biological product, wherein the reference drug product or the reference biological product is Hu 119-122. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a formulation different from a formulation of a reference drug product or reference biologic product, wherein the reference drug product or reference biologic product is Hu 119-122. OX40 agonist antibodies may be authorized by the drug regulatory agency (e.g., FDA and/or EMA of the european union in the united states). In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is Hu 119-122. In some embodiments, the biosimilar is provided in the form of a composition further comprising one or more excipients, wherein the one or more excipients are the same as or different from the excipients included in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is Hu 119-122.

Table 13: amino acid sequence of OX40 agonist antibody related to Hu119-122

In some embodiments, the OX40 agonist is Hu106-222, a humanized antibody commercially available from GlaxoSmithKline plc. The preparation and properties of Hu106-222 are described in U.S. patent nos. 9,006,399 and 9,163,085 and international patent publication No. WO2012/027328, the disclosures of which are incorporated herein by reference. The amino acid sequence of Hu106-222 is set forth in Table 14.

In one embodiment, an OX40 agonist comprises the heavy and light chain CDRs or Variable Regions (VRs) of Hu 106-222. In one embodiment, the OX40 agonist heavy chain variable region (V)_H) Comprises the amino acid sequence of SEQ ID NO: 94, OX40 agonist light chain variable region (V)_L) Comprises the amino acid sequence of SEQ ID NO: 95, and conservative amino acid substitutions thereof. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 94 and SEQ ID NO: 95 has at least 99% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 94 and SEQ IDNO: 95 has at least 98% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises a peptide that binds to seq id NOs: 94 and SEQ ID NO: 95 has at least 97% of the same V _HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 94 and SEQ ID NO: 95 has at least 96% of the same V_HAnd V_LAnd (4) a zone. In one embodiment, the OX40 agonist comprises an amino acid sequence that is identical to SEQ ID NO: 94 and SEQ ID NO: 95 has at least 95% of the same V_HAnd V_LAnd (4) a zone.

In one embodiment, the OX40 agonist comprises a polypeptide having the amino acid sequence of SEQ ID NO: 96, SEQ ID NO: 97 and SEQ ID NO: 98 and conservative amino acid substitutions thereof, and heavy chain CDR1, CDR2, and CDR3 domains having the sequences shown in SEQ ID NOs: 99, SEQ ID NO: 100 and SEQ ID NO: 101 and conservative amino acid substitutions thereof, and light chain CDR1, CDR2, and CDR3 domains thereof.

In one embodiment, the OX40 agonist is an OX40 agonist biosimilar monoclonal antibody approved by the drug regulatory agency with reference to Hu 106-222. In one embodiment, the biosimilar monoclonal antibody comprises an OX40 antibody comprising an amino acid sequence having at least 97% sequence identity (e.g., 97%, 98%, 99%, or 100% sequence identity) to an amino acid sequence of a reference drug product or a reference biological product and comprising one or more post-translational modifications as compared to the reference drug product or the reference biological product, wherein the reference drug product or the reference biological product is Hu 106-222. In some embodiments, the one or more post-translational modifications are selected from one or more of glycosylation, oxidation, deamidation, and truncation. In some embodiments, the biosimilar is an authorized or submitted authorized OX40 agonist antibody, wherein the OX40 agonist antibody is provided in a different formulation than a reference drug product or reference bioproduct, wherein the reference drug product or reference bioproduct is Hu 106-222. OX40 agonist antibodies may be authorized by the drug regulatory agency (e.g., FDA and/or EMA of the european union in the united states). In some embodiments, the biosimilar is provided in the form of a composition that further comprises one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is Hu 106-222. In some embodiments, the biosimilar is provided in the form of a composition that further comprises one or more excipients, wherein the one or more excipients are the same as or different from the excipients contained in a reference pharmaceutical product or a reference biological product, wherein the reference pharmaceutical product or the reference biological product is Hu 106-222.

Table 14: amino acid sequence of OX40 agonist antibody related to Hu106-222

In some embodiments, the OX40 agonist antibody is MEDI6469 (also referred to as 9B 12). MEDI6469 is a murine monoclonal antibody. Weinberg et al, J.Immunother.2006, 29, 575-. In some embodiments, the OX40 agonist is an antibody produced by the 9B12 hybridoma deposited by Biovest inc. (Malvern, MA, usa), as described in Weinberg et al, j.immunother.2006, 29, 575-. In some embodiments, the antibody comprises a CDR sequence of MEDI 6469. In some embodiments, the antibody comprises a heavy chain variable region sequence and/or a light chain variable region sequence of MEDI 6469.

In one embodiment, the OX40 agonist is L106 BD (Pharmingen product # 340420). In some embodiments, the OX40 agonist comprises the CDRs of antibody L106(BD Pharmingen product # 340420). In some embodiments, the OX40 agonist comprises a heavy chain variable region sequence and/or a light chain variable region sequence of antibody L106(BD Pharmingen product # 340420). In one embodiment, the OX40 agonist is ACT35(Santa Cruz Biotechnology, catalog No. 20073). In some embodiments, the OX40 agonist comprises the CDRs of antibody ACT35(Santa Cruz Biotechnology, catalog No. 20073). In some embodiments, the OX40 agonist comprises the heavy chain variable region sequence and/or the light chain variable region sequence of antibody ACT35(Santa cruz biotechnology, catalog No. 20073). In one embodiment, the OX40 agonist is a murine monoclonal antibody anti-mCD 134/mOX40 (clone OX86), commercially available from InVivoMAb, bioxcell inc, West Lebanon, NH.

In one embodiment, the OX40 agonist is selected from international patent application publication nos. WO95/12673, WO95/21925, WO2006/121810, WO2012/027328, WO2013/028231, WO2013/038191, and WO 2014/148895; european patent application EP 0672141; U.S. patent application publication nos. US2010/136030, US2014/377284, US2015/190506, and US2015/132288 (including clones 20E5 and 12H 3); and OX40 agonists described in U.S. patent nos. 7,504,101, 7,550,140, 7,622,444, 7,696,175, 7,960,515, 7,961,515, 8,133,983, 9,006,399, and 9,163,085, the disclosures of each of which are incorporated herein by reference in their entirety.

In one embodiment, the OX40 agonist is an OX40 agonist fusion protein as described in structure I-A (C-terminal Fc antibody fragment fusion protein) or structure I-B (N-terminal Fc antibody fragment fusion protein), or a fragment, derivative, conjugate, variant, or biological analog thereof. The properties of structures I-A and I-B are as described above and described in U.S. Pat. Nos. 9,359,420, 9,340,599, 8,921,519 and 8,450,460, the disclosures of which are incorporated herein by reference. Table 6 gives the amino acid sequence of the polypeptide domain of structure I-A. The Fc domain preferably comprises the entire constant domain (amino acids 17-230 of SEQ ID NO: 31), the entire hinge domain (amino acids 1-16 of SEQ ID NO: 31) or a portion of the hinge domain (e.g., amino acids 4-16 of SEQ ID NO: 31). Preferred linkers for linking C-terminal Fc antibodies may be selected from SEQ ID NOs: 32 to SEQ ID NO: 41, comprising a linker suitable for fusion to another polypeptide. Similarly, the amino acid sequence of the polypeptide domain of structure I-B is given in Table 7. If the Fc antibody fragment is fused to the N-terminus of a TNRFSF fusion protein (as in structures I-B), the sequence of the Fc module is preferably SED ID NO: 42, and the linker sequence is preferably selected from the group consisting of SED ID NO: 43 to SEQ ID NO: 45 are shown.

In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain selected from the group consisting of: the variable heavy and variable light chain of taliximab, the variable heavy and variable light chain of 11D4, the variable heavy and variable light chain of 18D8, the variable heavy and variable light chain of Hu119-122, the variable heavy and variable light chain of Hu106-222, the variable heavy and variable light chain selected from the variable heavy and variable light chain set forth in table 15, any combination of the foregoing variable heavy and variable light chain, and fragments, derivatives, conjugates, variants, and biological analogs thereof.

In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain comprising an OX40L sequence. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises one or more amino acid sequences comprising the sequence SEQ ID NO: 102, OX40 binding domain. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain comprising a soluble OX40L sequence. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises one or more amino acid sequences comprising the sequence of SEQ ID NO: 103, OX40 binding domain. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises one or more amino acid sequences comprising the sequence SEQ ID NO: 104 OX40 binding domain.

In one embodiment, an OX40 agonist fusion protein according to structure 1A or 1B comprises more than one OX40 binding domain that is V-containing 40 binding domain_HAnd V_LscFv domain of region V_HAnd V_LThe regions are respectively identical to SEQ ID NO: NO: 58 and SEQ ID NO: 59, wherein V is at least 95% identical_HAnd V_LThe domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain that is F-containing with the OX40 binding domain_HAnd V_LscFv domain of region V_HAnd V_LThe regions are respectively identical to SEQ ID NO: 68 and SEQ ID NO: 69, wherein V is at least 95% identical_HAnd V_LThe domains are connected by a linker. In one embodiment, OX40 agonist fusion proteins according to structure I-A or I-BComprising more than one OX40 binding domain, the OX40 binding domain being V-containing_HAnd V_LscFv domain of region V_HAnd V_LThe regions are respectively identical to SEQ ID NO: 78 and SEQ ID NO: 79 are at least 95% identical, wherein V_HAnd V_LThe domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain that is F-containing with the OX40 binding domain _HAnd V_LscFv domain of region V_HAnd V_LThe regions are respectively identical to SEQ ID NO: 86 and SEQ ID NO: 87 has at least 95% identity, wherein V_HAnd V_LThe domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain, the OX40 binding domain is V-comprising_HAnd V_LscFv domain of region V_HAnd V_LThe regions are respectively identical to SEQ ID NO: 94 and SEQ ID NO: 95, wherein V is at least 95% identical_HAnd V_LThe domains are connected by a linker. In one embodiment, an OX40 agonist fusion protein according to structure I-A or I-B comprises more than one OX40 binding domain that is F-containing with the OX40 binding domain_HAnd V_LscFv domain of region V_HAnd V_LZone and V shown in Table 15_HAnd V_LThe sequences have at least 95% identity, wherein V_HAnd V_LThe domains are connected by a linker.

Table 15: other polypeptide domains useful as OX40 binding domains (e.g., structures I-A and I-B) in fusion proteins or as scFv OX40 agonist antibodies

In one embodiment, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain further comprising an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain. In one embodiment, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising (i) a first soluble OX40 binding domain, (ii) a first peptide linker, (iii) a second soluble OX40 binding domain, (iv) a second peptide linker, and (v) a third soluble OX40 binding domain, further comprising an additional domain at the N-terminus and/or C-terminus, wherein the additional domain is a Fab or Fc fragment domain, wherein each soluble OX40 binding domain lacks a stem region (facilitates trimerization and provides a distance to the cell membrane, but is not part of the OX40 binding domain), and the first peptide linker and the second peptide linker independently have a length of 3 to 8 amino acids.

In one embodiment, the OX40 agonist is an OX40 agonist single chain fusion polypeptide comprising (i) a first soluble Tumor Necrosis Factor (TNF) superfamily cytokine domain, (ii) a first peptide linker, (iii) a second soluble TNF superfamily cytokine domain, (iv) a second peptide linker, and (v) a third soluble TNF superfamily cytokine domain, wherein each soluble TNF superfamily cytokine domain lacks a stem region, and the first and second peptide linkers independently have a length of 3 to 8 amino acids, wherein the TNF superfamily cytokine domain is an OX40 binding domain.

In some embodiments, the OX40 agonist is MEDI 6383. MEDI6383 is an OX40 agonist fusion protein, which can be prepared as described in U.S. patent No. 6,312,700, the disclosure of which is incorporated herein by reference.

In one embodiment, the OX40 agonist is an OX40 agonist scFv antibody comprising an antibody that binds to any of the foregoing V_LAny of the foregoing V linked by domains_HA domain.

In one embodiment, the OX40 agonist is the Cretive Biolabs OX40 agonist monoclonal antibody MOM-18455, commercially available from Cretive Biolabs, Inc. of Shirley, NY, USA.

In one embodiment, the OX40 agonist is the OX40 agonist antibody clone, Ber-ACT35, commercially available from BioLegend, Inc.

H. Alternative cell viability assays

Alternatively, cell viability assays can be performed after the first expansion of step B using standard assays known in the art. Alternatively, cell viability assays can be performed after the first expansion (sometimes referred to as initial bulk expansion) using standard assays known in the art. For example, a trypan blue exclusion assay can be performed on a large number of TIL samples, which selectively labels dead cells and allows viability assessment. Other assays for detecting viability may include, but are not limited to, Alamar blue assays; and MTT analysis.

1. Cell counting, viability, flow cytometry

In some embodiments, cell count and/or viability is measured. Expression of markers (such as, but not limited to, CD3, CD4, CD8, and CD56), as well as any other marker disclosed or described herein, can be measured by flow cytometry with an antibody, such as, but not limited to, using facscan to^TMFlow cytometers (BD Biosciences) are those commercially available from BD Bio-Sciences (BDbiosciences, San Jose, Calif.). Cells can be counted manually using a disposable c-chip hemocytometer (VWR, Batavia, IL), and viability can be assessed using any method known in the art, including but not limited to trypan blue staining.

In some cases, a large population of TILs may be cryopreserved immediately using the protocol described below. Alternatively, a large population of TIL may be REP as follows, and then cryopreserved. Similarly, where a genetically modified TIL is used for therapy, the bulk or REP TIL population may be genetically modified for appropriate therapy.

2. Cell culture

In one embodiment, a method for expanding TIL may comprise using about 5,000mL to about 25,000mL of cell culture medium, about 5,000mL to about 10,000mL of cell culture medium, or about 5,800mL to about 8,700mL of cell culture medium. In one embodiment, no more than one type of cell culture medium is used to expand the amount of TIL. Any suitable cell culture medium may be used, for example AIM-V cell culture medium (L-glutamine, 50. mu.M streptomycin sulfate and 10. mu.M gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this regard, the methods of the present invention advantageously reduce the amount of media and the number of types of media required to expand the amount of TIL. In one embodiment, expanding the amount of TIL may comprise adding fresh cell culture medium (also referred to as feeder cells) to the cells at a frequency of no more than once every three or once every four days. Expanding the number of cells with a vented container simplifies the steps required to expand the number of cells by reducing the feeding frequency required to expand the cells.

In one embodiment, the cell culture medium in the first gas-permeable container and/or the second gas-permeable container is unfiltered. The use of unfiltered cell culture medium can simplify the steps required to expand the cell number. In one embodiment, the cell culture medium in the first gas-permeable container and/or the second gas-permeable container is free of beta-mercaptoethanol (BME).

In one embodiment, the duration of the process is from about 14 days to about 42 days (e.g., about 28 days), the process comprising: obtaining a tumor tissue sample from a mammal; culturing a tumor tissue sample in a first gas-permeable container having a cell culture medium therein; obtaining TIL from a tumor tissue sample; the amount of TIL is expanded using aapcs in a second gas-permeable container with cell culture medium therein.

In one embodiment, the TIL is amplified in a gas permeable container. The TIL is amplified with PBMC using methods, compositions and devices known in the art, including those described in U.S. patent application publication No. US2005/0106717A1, the disclosure of which is incorporated herein by reference, using a gas permeable container. In one embodiment, the TIL is amplified in a gas permeable bag. In one embodiment, the TIL is expanded using a Cell Expansion System that expands the TIL in a gas permeable bag, such as the Xuri Cell Expansion System W25(Xuri Cell Expansion System W25) (GE Healthcare). In one embodiment, the TIL is expanded using a cell expansion system that expands TIL in an air-permeable bag, such as the WAVE bioreactor system, also known as Xuri cell expansion system W5(GE Healthcare). In one embodiment, the cell expansion system comprises a gas permeable cell pouch selected from the group consisting of: about 100mL, about 200mL, about 300mL, about 400mL, about 500mL, about 600mL, about 700mL, about 800mL, about 900mL, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, and about 10L.

In one embodiment, TIL may be amplified in a G-REX flask (commercially available from Wilson Wolf Manufacturing). Such embodiments allow for a population of cells from about 5X 10⁵Individual cell/cm²Amplification to 10X 10⁶To 30X 10⁶Individual cell/cm². In one embodiment, this expansion is performed without adding fresh cell culture medium (also known as feeder cells) to the cells. In one embodiment, no feed is required as long as the medium stays in the G-REX flask at a height of about 10 cm. In one embodiment, no feed is made but more than one cytokine is added. In one embodiment, the cytokine may be added as a bolus, without mixing the cytokine with the culture medium. Such containers, devices, and methods are known in the art and have been used to amplify TIL and include those described in U.S. patent application publication No. US2014/0377739a1, international patent application publication No. WO2014/210036a1, U.S. patent application publication No. US2013/0115617Al, international publication No. WO2013/188427a1, U.S. patent application publication No. US2011/0136228a1, U.S. patent application publication No. 8,809,050, international patent application publication No. WO 2011/0722015088 a2, U.S. patent application publication No. US2016/0208216a1, U.S. patent application publication No. US2012/0244133a1, international patent application publication No. WO2012/129201a1, U.S. patent application publication No. US2013/0102075a1, U.S. patent application publication No. 8,956,860, international patent application publication No. WO2013/173835a1, and U.S. patent application publication No. US/0175966 Al, the disclosures of which are incorporated herein by reference. Such methods are also described in Jin et al, J.I mmunitoperpy 2012, 35, 283-.

I. Genetic engineering of alternative TILs

In some embodiments, the TIL is optionally genetically engineered to include other functions, including but not limited to: high affinity T Cell Receptors (TCRs), such as TCRs that target tumor associated antigens (e.g., MAGE, HER2, or NY-ESO-1); or a Chimeric Antigen Receptor (CAR) that binds to a tumor-associated cell surface molecule (e.g., mesothelin) or a lineage restricted cell surface molecule (e.g., CD 19).

J. Optional cryopreservation of TIL

As described above and illustrated in steps a through E provided in fig. 8, cryopreservation can occur at many points throughout the TIL population expansion process. In some embodiments, the amplified TIL population may be cryopreserved after a second amplification (e.g., according to step D of fig. 8). Typically, TIL populations can be cryopreserved by placing them in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution were placed in a cryovial for 24 hours at-80 ℃ and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al, Acta Oncology 2013,52, 978-. In some embodiments, the TIL is cryopreserved in 5% DMSO. In some embodiments, TILs are cryopreserved in cell culture medium supplemented with 5% DMSO. In some embodiments, the TIL is cryopreserved according to the methods provided in examples 8 and 9.

When appropriate, the cells were removed from the freezer and thawed in a 37 ℃ water bath until approximately 4/5 solution was thawed. The cells are typically resuspended in complete medium, optionally washed one or more times. In some embodiments, thawed TILs may be counted and viability assessed as known in the art.

K. Method for phenotypic characterization of amplified TILs

Production of granzyme B: granzyme B is another measure of the ability of TIL to kill target cells. The levels of granzyme B in the culture supernatants restimulated as described above were assessed using antibodies to CD3, CD28 and CD137/4-1BB using a human granzyme B DuoSet ELISA kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions. In some embodiments, a second or additional amplification of TIL (such as those described in step D of fig. 8, including TILs referred to as rep TILs) has increased production of granzyme B.

In some embodiments, telomere length can be used as a measure of cell viability and/or cell function. In some embodiments, the telomes of TILs produced by the invention are unexpectedly the same in telomere length as TILs produced using methods other than those described herein, including, for example, methods other than those described in fig. 8. Telomere length measurement: various methods have been used to measure telomere length in genomic DNA and cell preparations. Telomere Restriction Fragment (TRF) analysis is the gold standard for measuring telomere length (de Lange et al, 1990). However, the main limitation of TRF is the requirement of large amounts of DNA (1.5. mu.g). Two widely used techniques for measuring telomere length, fluorescence in situ hybridization (FISH; Agilent technologies, Santa Clara, Calif.), and quantitative PCR, can be used with the present invention.

In some embodiments, TIL health is measured by IFN- γ secretion. In some embodiments, IFN- γ secretion is indicative of active TIL. In some embodiments, a potency assay that produces IFN- γ is employed. IFN- γ production can be measured by determining the level of cytokine IFN- γ in TIL medium stimulated with CD3, CD28, and CD137/4-1BB antibodies. IFN- γ levels in these stimulated TIL media can be determined by measuring IFN- γ release.

In some embodiments, the cytotoxic potential of TIL to lyse target cells is assessed using a co-culture assay of TIL with the bioluminescent cell line P815 (clone G6), according to the bioluminescent redirect lysis assay (titer assay) of the TIL assay, which measures the cytotoxicity of TIL in a highly sensitive dose-dependent manner.

In some embodiments, the present methods provide assays for assessing TIL viability using the methods described above. In some embodiments, the TIL is amplified as described above (including, e.g., as described in fig. 8). In some embodiments, the TIL is cryopreserved prior to assessing viability. In some embodiments, viability assessment comprises thawing the TIL prior to performing the first amplification, the second amplification, and the additional second amplification. In some embodiments, the methods provide assays that assess cell proliferation, cytotoxicity, cell death, and/or other terms related to viability of a TIL population. Viability may be measured by any of the TIL metabolism assays described above, as well as any known methods of assessing cell viability known in the art. In some embodiments, the methods provide assays for assessing cell proliferation, cytotoxicity, cell death, and/or other terms associated with viability of TILs expanded using the methods described herein, including the methods shown in fig. 8.

The invention also provides a determination method for determining the activity of the TIL. In some embodiments, the TIL has the same viability as a freshly harvested TIL and/or a TIL prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). In some embodiments, the TIL has increased viability as compared to freshly harvested TIL and/or TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). The present invention provides a method for determining Tumor Infiltrating Lymphocytes (TILs) activity by expanding TILs into a larger population of TILs, the method comprising:

(i) obtaining a first population of previously amplified TILs;

(ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population; and

(iii) performing a second expansion by supplementing the cell culture medium of the second TIL population with additional IL-2, OKT-3, and Antigen Presenting Cells (APCs), producing a third TIL population; wherein the number of third TIL groups is at least 100 times greater than the number of second TIL groups; wherein the second amplification is performed for at least 14 days to obtain a third population of TILs; wherein, the third TIL population viability was further analyzed.

In some embodiments, the method further comprises:

(iv) performing an additional second expansion by supplementing the cell culture medium of the third TIL population with additional IL-2, additional OKT-3, and additional APCs; wherein a further second amplification is carried out for at least 14 days to obtain a population of TILs that is larger than the population of TILs obtained in step (iii); wherein the viability of the third TIL population is further determined.

In some embodiments, the cells are cryopreserved prior to step (i).

In some embodiments, the cells are thawed prior to performing step (i).

In some embodiments, step (iv) is repeated 1 to 4 times to obtain sufficient TIL for analysis.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 40 days to about 50 days.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 42 days to about 48 days.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 42 days to about 45 days.

In some embodiments, steps (i) to (iii) or (iv) are performed for a period of about 44 days.

In some embodiments, the cells from step (iii) or step (iv) express similar levels of CD4, CD8, and TCR α β as freshly harvested cells.

In some embodiments, the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs).

In some embodiments, the PBMCs are added to the cell culture on any of days 9 to 17 of step (iii).

In some embodiments, the APC is an artificial APC (aapc).

In some embodiments, the method further comprises the step of transducing the first TIL population with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the method further comprises the step of transducing the first TIL population with an expression vector comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one intracellular domain of a T cell signaling molecule.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the viability of the TIL is determined.

In some embodiments, the viability of TIL is determined after cryopreservation.

In some embodiments, the viability of the TIL is determined after cryopreservation and after step (iv).

Multiple antigen receptors for T and B lymphocytes are produced by somatic recombination of a limited but large number of gene segments. These gene fragments: v (variable), D (variable), J (connecting), and C (constant) determine the binding specificity and downstream applications of immunoglobulins and T Cell Receptors (TCRs). The present invention provides methods of producing TILs that demonstrate and increase the diversity (sometimes referred to as polyclonality) of T cell banks. In some embodiments, the increase in diversity of the T cell bank is compared to freshly harvested TIL and/or TIL prepared using methods other than those provided herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the TILs obtained by the methods of the invention exhibit increased diversity in the T cell repertoire. In some embodiments, the TIL obtained from the first expansion exhibits an increase in diversity of the T cell pool. In some embodiments, the increase in diversity is an increase in immunoglobulin diversity and/or T cell receptor diversity. In some embodiments, the immunoglobulin diversity is immunoglobulin heavy chain diversity. In some embodiments, the immunoglobulin diversity is immunoglobulin light chain diversity. In some embodiments, the diversity is T cell receptor diversity. In some embodiments, the diversity is a diversity of T cell receptors selected from one of alpha, beta, gamma, and receptor. In some embodiments, expression of T Cell Receptors (TCR) alpha and/or beta is increased. In some embodiments, expression of T Cell Receptor (TCR) α is increased. In some embodiments, expression of T Cell Receptor (TCR) β is increased. In some embodiments, expression of TCRab (i.e., TCR α/β) is increased.

In accordance with the present disclosure, a method of determining TIL activity and/or further for administration to a subject. In some embodiments, a method of determining Tumor Infiltrating Lymphocytes (TILs) comprises:

(i) obtaining a first TIL population;

(ii) performing a first expansion by culturing the first TIL population in a cell culture medium comprising IL-2 and optionally OKT-3, to produce a second TIL population; and

(iv) harvesting, washing and cryopreserving a third TIL population;

(v) storing the cryopreserved TIL at a freezing temperature;

(vi) thawing the third TIL population to provide a thawed third TIL population;

(vii) subjecting a portion of the thawed third TIL population to a further second expansion by supplementing the cell culture medium of the third population with IL-2, OKT-3, and APC for a further expansion phase (sometimes referred to as a repp phase) of at least 3 days; performing a third amplification to obtain a fourth TIL group; comparing the number of TILs in the fourth TIL group with the number of TILs in the third TIL group to obtain a ratio;

(viii) (viii) determining whether the thawed TIL population is suitable for administration to a patient based on the ratio in step (vii);

(ix) when it is determined in step (viii) that the ratio of the number of TILs in the fourth TIL group to the number of TILs in the third TIL group is greater than 5: 1, administering a therapeutically effective dose of the thawed third TIL population to the patient.

In some embodiments, an additional amplification stage (sometimes referred to as a repp stage) is performed until the ratio of the number of TILs in the fourth TIL population to the number of TILs in the third TIL population is greater than 50: 1.

in some embodiments, the amount of TIL sufficient for a therapeutically effective dose is about 2.3 x 10¹⁰To about 13.7X 10¹⁰。

In some embodiments, steps (i) through (vii) are performed for a period of about 40 days to about 50 days. In some embodiments, steps (i) through (vii) are performed for a period of about 42 days to about 48 days. In some embodiments, steps (i) through (vii) are performed for a period of about 42 days to about 45 days. In some embodiments, steps (i) through (vii) are performed for a period of about 44 days.

In some embodiments, the cells from step (iii) or (vii) express similar levels of CD4, CD8, and TCR α β as freshly harvested cells. In some embodiments, the cell is a TIL.

In some embodiments, the antigen presenting cells are Peripheral Blood Mononuclear Cells (PBMCs). In some embodiments, the PBMCs are added to the cell culture on any of days 9 to 17 of step (iii).

In some embodiments, the APC is an artificial APC (aapc).

In some embodiments, the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a high affinity T cell receptor.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a Chimeric Antigen Receptor (CAR) comprising a single-chain variable fragment antibody fused to at least one intracellular domain of a T cell signaling molecule.

In some embodiments, the transduction step occurs before step (i).

In some embodiments, the viability of the TIL is determined after step (vii).

Other methods of determining TIL are also provided by the present disclosure. In some embodiments, the present disclosure provides a method of determining TIL, the method comprising:

(i) obtaining a portion of the cryopreserved first TIL population;

(ii) thawing the portion of the cryopreserved first TIL population;

(iii) Performing a first expansion by culturing the portion of the first TIL population in a cell culture medium comprising IL-2, OKT-3, and Antigen Presenting Cells (APCs) for an additional expansion phase (sometimes referred to as a repp phase) of at least 3 days, producing a second TIL population; wherein the portion of the first TIL group is compared to the second TIL group to obtain a ratio of TIL numbers; wherein a ratio of the number of TILs of the second group of TILs to the number of TILs of the portion of the first group of TILs is greater than 5: 1;

(iv) (iv) determining whether the first TIL population is suitable for therapeutic administration to the patient based on the ratio in step (iii);

(v) when it is determined in step (iv) that the ratio of the number of TILs in the second group of TILs to the number of TILs in the first group of TILs is greater than 5: 1, determining that the first TIL population is suitable for therapeutic administration.

In some embodiments, a ratio of the number of TILs in the second TIL group to the number of TILs of the portion of the first TIL group is greater than 50: 1.

in some embodiments, the method further comprises expanding the entire cryopreserved first TIL population of step (i) according to a method as described in any of the embodiments provided herein.

In some embodiments, the method further comprises administering the entire cryopreserved first TIL population from step (i) to the patient.

Closed system for TIL production

The present invention provides for the use of a closed system during the TIL cultivation process. Such closed systems may prevent and/or reduce microbial contamination, allow fewer flasks to be used, and may reduce costs. In some embodiments, the closure system uses two containers.

Such closed systems are well known in the art and can be found, for example, in http:// www.fda.gov/cber/guidelines. htm and https:// www.fda.gov/biologics Blood lipids/guidanceaceRegulatoryinformation/Guidances/Blood/ucm 076779. htm.

In some embodiments, the closure system comprises a luer lock and heat seal system as described in example 16. In some embodiments, the closed system is accessed through a syringe under sterile conditions to maintain sterility and closure of the system. In some embodiments, a closed system as described in example 16 is employed. In some embodiments, the TIL is formulated into a final product formulation container according to the method described in section 8.14 "final formulation and fill" of example 16.

Closed systems using aseptic methods are known and fully described, as described on the FDA website. See https:// www.fda.gov/biologics Blood vaccines/Guidances company regulated information/Guidances/Blood/ucm076779. htm.

Introduction to the design reside in

A sterile connection device (STCD) produces a sterile weld between two compatible tubes. This procedure allows for aseptic connection of various containers and tube diameters. The present guidelines describe recommended practices and procedures for using these devices. The present guidelines do not refer to data or information that the sterile connection equipment manufacturer must submit to the FDA for approval or approval for marketing. It is also important to note that the use of approved or authorized sterile connection equipment for unauthorized purposes in labels, according to Federal Food, Drug and Cosmetic Act, may result in the equipment being viewed as adulterated and branded.

In some embodiments, the closed system uses one container from the time tumor fragments are obtained until the TIL is ready for administration to a patient or cryopreservation. In some embodiments, when two containers are used, the first container is a closed G-container, and the TIL population is centrifuged and transferred to the infusion bag without opening the first closed G-container. In some embodiments, when two containers are used, the infusion bag is a hypo thermosol containing infusion bag. The closed system or closed TIL cell culture system is characterized in that once the tumor sample and/or tumor debris is added, the system is tightly sealed from the outside, creating a closed environment that is not invaded by bacteria, fungi and/or any other microbial contaminants.

In some embodiments, the microbial contamination is reduced by about 5% to about 100%. In some embodiments, the microbial contamination is reduced by about 5% to about 95%. In some embodiments, the microbial contamination is reduced by about 5% to about 90%. In some embodiments, the microbial contamination is reduced by about 10% to about 90%. In some embodiments, the microbial contamination is reduced by about 15% to about 85%. In some embodiments, the microbial contamination is reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%.

The closed system allows for growing TIL in the absence of microbial contamination and/or with a significant reduction in microbial contamination.

In addition, the pH, carbon dioxide partial pressure, and oxygen partial pressure of the TIL cell culture environment all vary with the cell culture. Therefore, even if a medium suitable for cell culture is circulated, it is necessary to constantly maintain the closed environment as an optimal environment for proliferation of TIL. For this reason, it is desirable to monitor physical factors of pH, partial pressure of carbon dioxide and partial pressure of oxygen in the culture solution of the closed environment by sensors whose signals are used to control a gas exchanger installed at an inlet of the culture environment, and to adjust the partial pressure of gas of the closed environment in real time according to the change of the culture solution to optimize the cell culture environment. In some embodiments, the present invention provides a closed cell culture system incorporating a gas exchanger equipped with a monitoring device at the inlet of the closed environment that measures the pH, partial pressure of carbon dioxide and partial pressure of oxygen of the closed environment and optimizes the cell culture environment by automatically adjusting the gas concentration according to signals from the monitoring device.

In some embodiments, the pressure in the enclosed environment is controlled continuously or intermittently. That is, for example, the pressure in the closed environment may be changed by the pressure maintaining means to ensure that the space is suitable for the growth of TIL in a positive pressure state, or to promote the exudation of fluid in a negative pressure state to promote cell proliferation. Furthermore, by intermittently applying the negative pressure, the circulating liquid in the closed environment can be uniformly and efficiently replaced by the temporary contraction of the volume of the closed environment.

In some embodiments, factors (e.g., IL-2 and/or OKT3 and combinations thereof) may be added in place of or in addition to optimal culture components for TIL propagation.

C. Cell culture

In an embodiment, a method of expanding TIL (including those described above and illustrated in FIG. 8) may comprise using about 5,000mL to about 25,000mL of cell culture medium, about 5,000mL to about 10,000mL of cell culture medium, or about 5,800mL to about 8,700mL of cell culture medium. In some embodiments, for example as described in example 21, the culture medium is a serum-free medium. In some embodiments, the medium in the first amplification is serum-free. In some embodiments, the medium in the second amplification is serum-free. In some embodiments, the medium in both the first and second amplifications is serum-free. In one embodiment, no more than one type of cell culture medium is used to expand the amount of TIL. Any suitable cell culture medium may be used, for example AIM-V cell culture medium (L-glutamine, 50. mu.M streptomycin sulfate and 10. mu.M gentamicin sulfate) cell culture medium (Invitrogen, Carlsbad CA). In this regard, the methods of the present invention advantageously reduce the amount of media and the number of types of media required to expand the number of TILs. In one embodiment, expanding the amount of TIL may comprise feeding the cells at a frequency of no more than once every three days or once every four days. Expanding the number of cells in a gas permeable container simplifies the procedure required to expand the number of cells by reducing the feeding frequency required to expand the cells.

In one embodiment, the cell culture medium in the first gas-permeable container and/or the second gas-permeable container is unfiltered. The use of unfiltered cell culture medium simplifies the procedure required to expand the cell number. In one embodiment, the cell culture medium in the first gas-permeable container and/or the second gas-permeable container is devoid of beta-mercaptoethanol (BME).

In one embodiment, the duration of the method is from about 7 days to 14 days (e.g., about 11 days), the method comprising: obtaining a tumor tissue sample from a mammal; culturing a tumor tissue sample in a first gas-permeable container having a cell culture medium therein; obtaining TIL from a tumor tissue sample; the amount of TIL is expanded in a second gas-permeable container containing cell culture medium. In some embodiments, pre-REP is from about 7 days to 14 days, e.g., about 11 days. In some embodiments, REP is from about 7 days to 14 days, for example about 11 days.

In one embodiment, the TIL is amplified in a gas permeable container. Gas permeable containers have been used to amplify TILs using PBMCs using methods, compositions and devices known in the art, including those described in U.S. patent application publication No. 2005/0106717A1, the disclosure of which is incorporated herein by reference. In one embodiment, the TIL is amplified in a gas permeable bag. In one embodiment, the TIL is expanded in a gas permeable bag using a cell expansion system that expands TIL, such as the Xuri cell expansion system W25 (GEHealthcare). In one embodiment, the TIL is amplified in an air permeable bag using a cell expansion system that amplifies TIL, such as a WAVE bioreactor system (also known as Xuri cell expansion system W5) (GE Healthcare). In one embodiment, the cell expansion system comprises a gas-permeable cell bag having a volume selected from the group consisting of: about 100mL, about 200mL, about 300mL, about 400mL, about 500mL, about 600mL, about 700mL, about 800mL, about 900mL, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, and about 10L.

In one embodiment, TIL may be amplified in a G-Rex flask (commercially available from Wilson Wolf Manufacturing). Such embodiments allow for a population of cells from about 5X 10⁵Individual cell/cm²Amplification was 10X 10⁶Individual cell/cm²To 30X 10⁶Individual cell/cm². In one embodiment, no feed is required. In one embodiment, no feed is required as long as the medium stays at a height of about 10cm in the G-Rex flask. In one embodiment, no feed is required, but more than one cytokine is added. In one embodiment, the cytokine may be added as a bolus injection without mixing the cytokine with the culture medium. Such containers, devices, and methods are known in the art and have been used to amplify TIL and include U.S. patent application publication No. US2014/0377739a1, international publication No. WO2014/210036a1, U.S. patent application publication No. US2013/0115617a1, international publication No. WO2013/188427a1, U.S. patent application publication No. US2011/0136228a1Those described in patent No. US8,809,050B2, international publication No. WO2011/072088a2, U.S. patent application publication No. US2016/0208216a1, U.S. patent application publication No. US2012/0244133a1, international publication No. WO2012/129201a1, U.S. patent application publication No. 20132013/0102075 a1, U.S. patent No. 8,956,860B2, international publication No. WO2013/173835a1, U.S. patent application publication No. US2015/0175966a1, the disclosures of which are incorporated herein by reference. Jin et al, j.immunotherpy, 2012, 35: 283- "292" also describes such processes.

D. Genetic engineering of alternative TILs

E. Optional cryopreservation of TIL

Alternatively, a large population of TILs or an expanded population of TILs may be cryopreserved. In some embodiments, the therapeutic TIL population is cryopreserved. In some embodiments, the TIL harvested after the second amplification is cryopreserved. In some embodiments, in the exemplary step F of fig. 8, the TIL is cryopreserved. In some embodiments, the TIL is cryopreserved in an infusion bag. In some embodiments, the TIL is cryopreserved prior to being placed in an infusion bag. In some embodiments, the TIL is cryopreserved and not placed in an infusion bag. In some embodiments, cryopreservation media is used for cryopreservation. In some embodiments, the cryopreservation media comprises dimethyl sulfoxide (DMSO). This is typically accomplished by placing the TIL population in a freezing solution, such as 85% complement inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution were placed in a cryopreservation tube and stored at-80 ℃ for 24 hours, and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al, Acta Oncology 2013, 52, 978-.

When appropriate, cells were removed from the refrigerator and thawed in a 37 ℃ water bath until approximately 4/5 solution was thawed. The cells are typically resuspended in complete medium and optionally washed one or more times. In some embodiments, thawed TILs may be counted and viability assessed as known in the art.

In a preferred embodiment, the TIL population is cryopreserved using CS10 cryopreservation medium (CryoStor 10, BioLifeSolutions). In a preferred embodiment, the TIL population is cryopreserved using a cryopreservation medium comprising dimethyl sulfoxide (DMSO). In a preferred embodiment, CS10 and 1: the TIL population was cryopreserved at a ratio of 1 (volume: volume). In a preferred embodiment, CS10 and about 10: 1 (volume: volume) ratio the TIL population was cryopreserved, and the cell culture medium further included additional IL-2.

As discussed above in steps a through E, cryopreservation may occur at many points throughout the TIL population expansion process.

As described above, and as illustrated in steps a through E provided in fig. 8, cryopreservation may occur at many time points throughout the TIL population amplification process. In some embodiments, the amplified TIL population after the second amplification can be cryopreserved (e.g., provided according to step D of panel a). Cryopreservation is typically accomplished by placing the TIL population in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution were placed in a cryopreservation tube and stored at-80 ℃ for 24 hours, and optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al, Acta Oncology 2013, 52, 978-. In some embodiments, TIL is cryopreserved in 5% DMSO. In some embodiments, the TIL is cryopreserved in cell culture medium plus 5% DMSO. In some embodiments, the TIL is cryopreserved according to the methods provided in example 16.

In some embodiments, a plurality of TIL populations after a first amplification according to step B or amplified TIL populations after one or more second amplifications according to step D may be cryopreserved. Typically, TIL populations can be cryopreserved by placing them in a freezing solution, such as 85% complement-inactivated AB serum and 15% dimethyl sulfoxide (DMSO). The cells in solution were placed in a cryovial and stored at-80 ℃ for 24 hours, optionally transferred to a gaseous nitrogen freezer for cryopreservation. See Sadeghi et al, Acta Oncology 2013,52, 978-.

In some cases, the TIL population of step B may be immediately cryopreserved using the protocol described below. Alternatively, step C and step D may be performed on a large population of TILs, followed by cryopreservation after step D. Similarly, where a genetically modified TIL is used for therapy, the TIL populations of step B or D steps may be genetically modified for appropriate therapy.

Method of treating a patient

The treatment method begins with the collection of initial TILs and the culture of TILs. These methods have been described in the art, for example, by Jin et al (J.Immunotherapy,2012,35 (3): 283-. Embodiments of the treatment methods are described in the entire section below, including the examples.

The amplified TILs produced according to the methods described herein, including, for example, as described in steps A through F above or as described in steps A through F above (e.g., as also shown in FIG. 8), find particular use in treating cancer patients (e.g., as described in Goff et al, J. clinical Oncology 2016, 34 (20): 2389-. In some embodiments, TIL is grown from excised metastatic melanoma deposits as previously described (see Dudley et al, J Immunother.,2003, 26: 332-. Fresh tumors can be dissected under sterile conditions. Representative samples can be collected for formal pathology analysis. Can use 2mm³To 3mm³Of the individual fragments of (a). In some embodimentsWherein 5, 10, 15, 20, 25 or 30 samples are obtained per patient. In some embodiments, 20, 25, or 30 samples are obtained per patient. In some embodiments, 20, 22, 24, 26, or 28 samples are obtained per patient. In some embodiments, 24 samples are obtained per patient. Samples can be placed in individual wells of a 24-well plate, maintained in growth medium with high doses of IL-2(6,000IU/mL), and monitored for tumor destruction and/or proliferation of TILs. Any tumor with remaining viable cells after processing can be enzymatically digested into a single cell suspension and cryopreserved as described herein.

In some embodiments, successfully grown TILs may be sampled for phenotypic analysis (CD3, CD4, CD8, and CD56) and detected against autologous tumors as appropriate. TIL can be considered reactive if the overnight co-culture produced interferon-gamma (IFN-. gamma.) levels > 200pg/mL and twice the background. (Goff et al, J Immunother, 2010, 33: 840-847; the entire contents of which are incorporated herein by reference in their entirety). In some embodiments, cultures with evidence of autoreactivity or sufficient growth patterns may be selected for a second amplification (e.g., a second amplification provided according to step D of fig. 8), including a second amplification sometimes referred to as rapid amplification (REP). In some embodiments, amplified TILs with high auto-reactivity (e.g., high proliferation during the second amplification) are selected for additional second amplifications. In some embodiments, TILs with high auto-reactivity (e.g., high proliferation during the second amplification as provided in step D of fig. 8) are selected for additional second amplifications according to step D of fig. 8.

In some embodiments, the patient does not directly perform ACT (adoptive cell transfer), e.g., in some embodiments, the cells are not used immediately after tumor harvesting and/or first expansion. In such embodiments, the TIL may be cryopreserved and thawed 2 days prior to administration to the patient. In such embodiments, the TIL may be cryopreserved and thawed 1 day prior to administration to the patient. In some embodiments, the TIL may be cryopreserved and thawed immediately prior to administration to a patient.

Cryopreserved samples of infusion bags TIL can be analyzed by flow cytometry (e.g., FlowJo) and by any of the methods described herein for the surface markers of cellular phenotype, CD3, CD4, CD8, CCR7, and CD45RA (BD BioSciences). Serum cytokines were measured by using standard enzyme-linked immunosorbent assay techniques. The increase in serum IFN-g is defined as ≧ 100pg/mL and greater than 43 baseline levels.

In some embodiments, TILs produced by the methods provided herein (e.g., those illustrated in fig. 8) provide a surprising improvement in the clinical efficacy of TILs. In some embodiments, TILs produced by methods provided herein (e.g., those shown in fig. 8) exhibit increased clinical efficacy as compared to TILs produced by methods other than those described herein (e.g., methods other than those shown in fig. 8). In some embodiments, methods other than those described herein include methods referred to as process 1C and/or first generation (first generation). In some embodiments, increased efficacy is measured by DCR, ORR, and/or other clinical responses. In some embodiments, TILs produced by methods provided herein (e.g., the TILs shown in fig. 8) exhibit similar response times and safety as TILs produced by methods other than those described herein (including, for example, methods other than those shown in fig. 8, e.g., the Gen 1 process).

In some embodiments, IFN-gamma (IFN- γ) is indicative of therapeutic efficacy and/or increased clinical efficacy. In some embodiments, IFN- γ in the blood of a subject treated with TIL is indicative of active TIL. In some embodiments, IFN- γ production titer assay is employed. IFN- γ production is another measure of cytotoxic potential. IFN- γ production can be measured by determining the level of the cytokine IFN- γ in the blood, serum, or TIL of a subject treated with a TIL prepared by the methods of the invention, including, for example, those shown in FIG. 8. In some embodiments, an increase in IFN- γ is indicative of therapeutic efficacy in a patient treated with a TIL produced by the methods of the invention. In some embodiments, IFN- γ is increased 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more as compared to untreated patients and/or as compared to patients treated with TILs prepared using methods other than those provided herein, including, for example, methods other than those described in fig. 8. In some embodiments, IFN- γ is increased by 1-fold compared to untreated patients and/or to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). In some embodiments, IFN- γ is increased 2-fold compared to untreated patients and/or patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). In some embodiments, IFN- γ is increased by a factor of 3 compared to untreated patients and/or to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). In some embodiments, IFN- γ is increased 4-fold compared to untreated patients and/or compared to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). In some embodiments, IFN- γ is increased 5-fold compared to untreated patients and/or to patients treated with TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). In some embodiments, IFN- γ is measured using a QuantikineELISA kit. In some embodiments, IFN- γ is measured in a subject treated with a TIL ex vivo for a TIL prepared by the methods of the invention (including, e.g., those described in fig. 8). In some embodiments, IFN- γ is measured in the blood of a subject treated with a TIL prepared by the methods of the invention (including, e.g., those described in fig. 8). In some embodiments, IFN- γ is measured in serum of a subject treated with a TIL prepared by the methods of the invention (including, e.g., those described in fig. 8).

In some embodiments, TILs prepared by the methods of the invention (including, for example, those described in fig. 8) exhibit increased polyclonality as compared to TILs produced by other methods (including methods other than those shown in fig. 8, e.g., a method referred to as the process 1C process). In some embodiments, significantly improved polyclonality and/or increased polyclonality is indicative of an increase in therapeutic and/or clinical efficacy. In some embodiments, polyclonality refers to T cell bank diversity. In some embodiments, an increase in polyclonality may indicate therapeutic efficacy with respect to administering the TILs produced by the methods of the invention. In some embodiments, the polyclonality is increased 1-fold, 2-fold, 10-fold, 100-fold, 500-fold, or 1000-fold compared to TILs prepared using methods other than those provided herein (including, e.g., methods other than those described in fig. 8). In some embodiments, polyclonality is increased by 1-fold as compared to TILs prepared using methods other than those provided herein (including, e.g., methods other than those described in fig. 8). In some embodiments, polyclonality is increased by 2-fold as compared to TILs prepared using methods other than those provided herein (including, e.g., methods other than those described in fig. 8). In some embodiments, the polyclonality is increased by 10-fold compared to TILs prepared using methods other than those provided herein (including, e.g., methods other than those described in fig. 8). In some embodiments, polyclonality is increased 100-fold as compared to TILs prepared using methods other than those provided herein (including, e.g., methods other than those described in fig. 8). In some embodiments, polyclonality is increased 500-fold compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8). In some embodiments, polyclonality is increased 1000-fold compared to TILs prepared using methods other than those provided herein (including, for example, methods other than those described in fig. 8).

As known in the art and as described herein, measures of efficacy may include Disease Control Rate (DCR) and Overall Response Rate (ORR).

1. Methods of treating cancer and other diseases

The compositions and methods described herein are useful in methods of treating diseases. In one embodiment, they are used to treat hyperproliferative diseases. They may also be used to treat other diseases described herein and in the following paragraphs.

In some embodiments, the hyperproliferative disease is cancer. In some embodiments, the hyperproliferative disease is a solid tumor cancer. In some embodiments, the solid tumor cancer is selected from: melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer including Head and Neck Squamous Cell Carcinoma (HNSCC), renal cancer, and renal epithelial renal cell carcinoma. In some embodiments, the hyperproliferative disease is a hematologic malignancy. In some embodiments, the solid tumor cancer is selected from chronic lymphocytic leukemia, acute lymphocytic leukemia, diffuse large B-cell lymphoma, non-hodgkin's lymphoma, follicular lymphoma, and mantle cell lymphoma.

In one embodiment, the invention includes a method of treating cancer with a population of TILs, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to infusion of TILs in accordance with the present disclosure. In one embodiment, the non-myeloablative treatment is administration of cyclophosphamide at 60 mg/kg/day for 2 days (27 and 26 days prior to TIL infusion) and at 25mg/m²Fludarabine was administered daily for 5 days (27 to 23 days before TIL infusion). In one embodiment, following non-myeloablative treatment and TIL infusion (on day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 at 720,000IU/kg every 8 hours until physiologically tolerated.

The compounds and combinations of compounds described herein can be tested for efficacy in the treatment, prevention and/or management of the indicated disease or condition using various models known in the art, which provide guidance for the treatment of human diseases. For example, models for determining the efficacy of ovarian cancer treatment are described, e.g., in mulleny et al, Endocrinology 2012, 153, 1585-92; and Fong et al, j.ovarian res.2009, 2, 12. Models for determining the therapeutic efficacy of pancreatic cancer are described in Herreros-Villanueva et al, World j. gastroenterol 2012, 18, 1286-1294. Models for determining the efficacy of Breast Cancer treatment are described, for example, in Fantozzi, Breast Cancer res.2006, 8, 212. Models for determining the efficacy of Melanoma treatment are described, for example, in Damsky et al, Pigment Cell & Melanoma Res.2010,23, 853-. Models for determining the efficacy of lung cancer treatment are described, for example, in Meuwissen et al, Genes & Development,2005,19, 643-664. Models for determining the efficacy of lung cancer treatment are described, for example, in Kim, clin. exp. otorhinolaryngol.2009,2, 55-60; and Sano, Head neckoncol.2009,1, 32.

In some embodiments, IFN-gamma (IFN- γ) is indicative of therapeutic efficacy of a treatment for a hyperproliferative disease. In some embodiments, IFN- γ in the blood of a subject treated with TIL is indicative of active TIL. In some embodiments, IFN-gamma production titer assay is used. IFN- γ production is another measure of cytotoxic potential. Production of IFN- γ can be measured by determining the level of the cytokine IFN- γ in the blood of subjects treated with TILs produced by the methods of the present invention, including, for example, those described in FIG. 8. In some embodiments, the TIL obtained by the methods of the invention provides an increase in IFN- γ in the blood of a subject treated with a TIL by the methods of the invention, as shown in fig. 13, as compared to a subject treated with a TIL prepared using a method referred to as process 1C. In some embodiments, an increase in IFN- γ is indicative of therapeutic efficacy in a patient treated with a TIL produced by the methods of the invention. In some embodiments, IFN- γ is increased 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared by a method other than the methods described herein (including, e.g., a method other than that shown in fig. 8). In some embodiments, IFN- γ is increased by a factor of 1 compared to untreated patients and/or to patients treated with TILs prepared by methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, IFN- γ is increased 2-fold compared to untreated patients and/or compared to patients treated with TILs prepared by methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, IFN- γ is increased by a factor of 3 compared to untreated patients and/or to patients treated with TILs prepared by methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, IFN- γ is increased 4-fold as compared to an untreated patient and/or as compared to a patient treated with a TIL prepared by a method other than the methods described herein (including, for example, a method other than that shown in fig. 8). In some embodiments, IFN- γ is increased 5-fold as compared to untreated patients and/or as compared to patients treated with TILs prepared by methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the Quantikine ELISA kit is used to measure IFN- γ. In some embodiments, the Quantikine ELISA kit is used to measure IFN- γ. In some embodiments, IFN- γ is measured in a patient's ex vivo TIL treated with a TIL produced by the methods of the invention. In some embodiments, IFN- γ is measured in the blood of a patient treated with a TIL produced by the methods of the invention. In some embodiments, IFN- γ is measured in the serum of a patient treated with TIL produced by the methods of the invention.

In some embodiments, TILs prepared by the methods of the invention (including, for example, those described in fig. 8) exhibit increased polyclonality as compared to TILs produced by other methods (other than those shown in fig. 8, e.g., a method referred to as the process 1C process). In some embodiments, a significantly improved polyclonality and/or increased polyclonality indicates an increased therapeutic and/or clinical efficacy of the cancer treatment. In some embodiments, polyclonality refers to T cell bank diversity. In some embodiments, an increase in polyclonality may indicate therapeutic efficacy with respect to administering the TILs produced by the methods of the invention. In some embodiments, the polyclonality is increased 1-fold, 2-fold, 10-fold, 100-fold, 500-fold, or 1000-fold compared to TILs prepared using methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the polyclonality is increased 1-fold compared to untreated patients and/or patients treated by TILs prepared using methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the polyclonality is increased 2-fold compared to untreated patients and/or patients treated by TILs prepared using methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the polyclonality is increased 10-fold compared to untreated patients and/or patients treated by TILs prepared using methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the polyclonality is increased 100-fold compared to untreated patients and/or patients treated by TILs prepared using methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the polyclonality is increased 500-fold compared to untreated patients and/or patients treated by TILs prepared using methods other than those described herein (including, for example, methods other than those shown in fig. 8). In some embodiments, the polyclonality is increased 1000-fold compared to untreated patients and/or patients treated by TILs prepared using methods other than those described herein (including, for example, methods other than those shown in fig. 8).

2. Method of co-administration

In some embodiments, TILs produced as described herein, including, for example, TILs derived from the methods described in steps a through F of fig. 8, may be administered in combination with one or more immune checkpoint modulators, such as antibodies described below. For example, antibodies that target PD-1 and that can be co-administered with TILs of the invention include, for example, but are not limited to, nivolumab (BMS-936558, Bristol-Myers Squibb;

) Pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck;

) Humanized anti-PD-1 antibody JS001(Shanghai JunShi), monoclonal anti-PD-1 antibody TSR-042(Tesaro, Inc.), Pidilizumab (anti-PD-1 mAb CT-011, Medvation), anti-PD-1 monoclonal antibody BGB-A317(BeiGene) and/or anti-PD-1 antibody SHR-1210(Shanghai HengRui), human monoclonal antibody REGN2810(Regeneron), human monoclonal antibody MDX-1106(Bristol-Myers Squibb), and/or human monoclonal antibody MDX-1106(Bristol-Myers Squibb)The humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is from a clone: RMP1-14 (rat IgG) -BioXcell catalog number BP 0146. Other suitable antibodies suitable for use in the method of co-administration of TILs produced according to steps a through F described herein are anti-PD-1 antibodies disclosed in U.S. patent No. 8,008,449, which is incorporated herein by reference. In some embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibody known in the art that binds to PD-L1 and disrupts the interaction between PD-1 and PD-L1 and stimulates an anti-tumor immune response is suitable for use in the method of co-administration of TILs produced according to steps a through F described herein. For example, antibodies that target PD-L1 and are in clinical trials include BMS-936559(Bristol-Myers Squibb) and MPDL3280A (Genentech). Other suitable antibodies targeting PD-L1 are disclosed in U.S. patent No. 7,943,743, which is incorporated herein by reference. One of ordinary skill in the art will appreciate that any antibody that binds to PD-1 or PD-L1, disrupts PD-1/PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the method of co-administration of TILs produced according to steps a through F described herein. In some embodiments, a subject administered a TIL combination produced according to steps a through F is co-administered with an anti-PD-1 antibody when the patient has a cancer type that is refractory to treatment with only the anti-PD-1 antibody. In some embodiments, the TIL is administered to the patient in combination with anti-PD-1 when the patient has reconstituted melanoma (refactory melanoma). In some embodiments, when the patient has non-small cell lung cancer (NSCLC), the TIL is administered to the patient in combination with anti-PD-1.

3. Alternative patient lymphocyte depletion pretreatment

In one embodiment, the invention includes a method of treating cancer with a population of TILs, wherein, according to the present disclosure, a patient is pre-treated with non-myeloablative chemotherapy prior to infusion of TILs. In one embodiment, the invention includes cancer in a patient with a TIL population who has been previously treated with non-myeloablative chemotherapy. In one embodiment, the TIL population is administered by infusion. In one embodiment, non-myeloablativeThe treatment was 60 mg/kg/day cyclophosphamide for 2 days (day 27 and 26 before TIL infusion) and 25mg/m fludarabine²Day, for 5 days (27 to 23 days before TIL infusion). In one embodiment, following non-myeloablative chemotherapy and TIL infusion according to the present disclosure (day 0), patients receive intravenous infusion of IL-2 (aldesleukin, commercially available as PROLEUKIN) to physiological tolerance every 8 hours at 720,000IU/kg intravenously. In certain embodiments, the TIL population is used in combination with IL-2 for the treatment of cancer, wherein IL-2 is administered after the TIL population.

Experimental results indicate that lymphocyte depletion prior to adoptive transfer of tumor-specific T lymphocytes plays a key role in improving therapeutic efficacy by eliminating the competing elements of regulatory T cells and the immune system ("cytokine deposition"). Thus, some embodiments of the invention employ a lymphocyte depletion step (sometimes also referred to as "immunosuppression modulation") on the patient prior to introducing the TILs of the invention.

Typically, lymphocyte depletion is achieved by administration of fludarabine or cyclophosphamide (the active form is known as macsfamide) and combinations thereof. Such methods are described in Gassner et al, Cancer I mmunol I mmunother.2011, 60, 75-85; muranski et al, nat. clin. pract. oncol., 2006, 3, 668-one 681, Dudley, etc.; j.clin.oncol.2008, 26, 5233-.

In some embodiments, the fludarabine is administered at a concentration of 0.5 μ g/mL to 10 μ g/mL fludarabine. In some embodiments, the fludarabine is administered at a concentration of 1 μ g/mL fludarabine. In some embodiments, the fludarabine treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or longer. In some embodiments, fludarabine is administered at a dose of 10 mg/kg/day, 15 mg/kg/day, 20 mg/kg/day, 25 mg/kg/day, 30 mg/kg/day, 35 mg/kg/day, 40 mg/kg/day, or 45 mg/kg/day. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, fludarabine treatment is administered at 35 mg/kg/day for 4 to 5 days. In some embodiments, fludarabine treatment is administered at 25 mg/kg/day for 4 to 5 days.

In some embodiments, a concentration of 0.5 to 10 μ g/mL of cyclophosphamide (the active form of cyclophosphamide) is obtained by administration of cyclophosphamide. In some embodiments, a concentration of 1 μ g/mL of cyclophosphamide (the active form of cyclophosphamide) is obtained by administration of cyclophosphamide. In some embodiments, cyclophosphamide treatment is administered for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days or longer. In some embodiments, cyclophosphamide is present at 100mg/m ²150 mg/m/day²175 mg/m/day ²200 mg/m/day²225 mg/m/day²250 mg/m/day²275 mg/m/day²Daily or 300mg/m²Dose per day. In some embodiments, cyclophosphamide is administered intravenously (i.e., i.v.). In some embodiments, cyclophosphamide treatment is administered at 35 mg/kg/day for 2 to 7 days. In some embodiments, cyclophosphamide treatment is at 250mg/m²Daily intravenous administration was for 4 to 5 days. In some embodiments, cyclophosphamide treatment is at 250mg/m²Intravenous administration was given for 4 days.

In some embodiments, lymphocyte depletion is performed by administering fludarabine and cyclophosphamide together to a patient. In some embodiments, the fludarabine is at 25mg/m²Administered intravenously daily and cyclophosphamide at 250mg/m ²Intravenous administration was given for 4 days.

In one embodiment, the concentration is controlled by adding 60mg/m²Cyclophosphamide was administered at a dose of one day for 2 days, then 25mg/m²The dose of fludarabine/day was administered for 5 days for lymphocyte depletion.

Scheme for IL-2

In one embodiment, the IL-2 regimen comprises a high dose IL-2 regimen, wherein the high dose IL-2 regimen comprises aclidinin or a biological analog or variant thereof administered intravenously starting the second day after administration of the therapeutically effective dose of the therapeutic TIL population, wherein the aclidinin or a biological analog or variant thereof is administered at a dose of 0.037mg/kg or 0.044mg/kg (patient body weight) of intravenous bolus injection at 15 minutes every 8 hours until tolerated for a maximum of 14 doses. After 9 days of rest, this schedule can be repeated for 14 more doses, for a total of up to 28 doses.

In one embodiment, the IL-2 regimen comprises a decreasing IL-2 regimen. Decreasing IL-2 protocols are described in O' Day et al, J.Clin.Oncol.1999,17,2752-61 and Eton et al, Cancer 2000,88,1703-9, the disclosures of which are incorporated herein by reference. In one embodiment, the reduced IL-2 regimen comprises intravenous administration of 18X 10 over 6 hours⁶IU/m²Followed by intravenous administration of 18X 10 over 12 hours ⁶IU/m²Then administered intravenously 18X 10 over 24 hours⁶IU/m²Then injected intravenously at 4.5X 10 times over 72 hours⁶IU/m². This treatment cycle may be repeated every 28 days for a maximum of 4 cycles. In one embodiment, the decreasing IL-2 regimen comprises 18,000,000IU/m on day 1²Comprising 9,000,000IU/m on day 2²Containing 4,500,000IU/m on days 3 and 4²。

In one embodiment, the IL-2 regimen comprises administering pegylated IL-2 at a dose of 0.10 mg/day to 50 mg/day every 1, 2, 4, 6, 7, 14, or 21 days.

5. Adoptive cell transfer

Adoptive Cell Transfer (ACT) is a very effective form of immunotherapy, involving the transfer of immune cells with anti-tumor activity into cancer patients. ACT is a therapeutic approach involving the in vitro identification of lymphocytes having anti-tumor activity, the massive in vitro expansion of these cells, and their injection into a host with cancer. Lymphocytes for adoptive transfer can be from the stroma of the resected tumor (tumor infiltrating lymphocytes or TILs). TILs for ACT may be prepared as described herein. In some embodiments, the TIL is prepared, for example, according to the method described in fig. 8. They may also be derived from or from blood if they are genetically engineered to express anti-tumor T Cell Receptors (TCRs) or Chimeric Antigen Receptors (CARs), enriched mixed lymphocyte tumor cell cultures (MLTCs), or use autologous antigen presenting cells and tumor-derived peptide clones. ACT in which lymphocytes are derived from a cancerous host to be infused is referred to as autologous ACT. U.S. publication No. 2011/0052530, which is incorporated by reference in its entirety, relates to methods for performing adoptive cell therapy to promote cancer regression, primarily for treating patients with metastatic melanoma. In some embodiments, the TIL may be administered as described herein. In some embodiments, the TIL may be administered in a single dose. Such administration may be by injection, for example, intravenous injection. In some embodiments, the TIL and/or cytotoxic lymphocytes may be administered in multiple doses. Administration may be once, twice, three times, four times, five times, six times or more than six times per year. Administration may be monthly, biweekly, weekly, or every other day. If desired, the administration of TIL and/or cytotoxic lymphocytes may be continued.

6. Exemplary therapeutic embodiments

In some embodiments, the present disclosure provides methods of treating cancer with a Tumor Infiltrating Lymphocyte (TIL) population, comprising the steps of: (a) obtaining a first TIL population from an excised tumor of a patient; (b) performing an initial expansion of the first TIL population in a first cell culture medium to obtain a second TIL population; wherein the number of second TIL groups is at least 5 times greater than the number of first TIL groups; wherein the first cell culture medium comprises IL-2; (c) rapidly expanding the second TIL population in a second cell culture medium using a myeloid artificial antigen presenting cell (myeloid aAPC) population to obtain a third TIL population; wherein 7 days after the rapid amplification, the number of the third population of TILs is at least 50-fold greater than the number of the second population of TILs; wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third TIL population to the cancer patient. In some embodiments, the present invention discloses a population of Tumor Infiltrating Lymphocytes (TILs) for use in treating cancer, wherein the TIL population is obtainable by a method comprising: (b) initially expanding a first TIL population obtained from a resected tumor of a patient in a first cell culture medium to obtain a second TIL population; wherein the number of second TIL groups is at least 5 times greater than the number of first TIL groups; wherein the first cell culture medium comprises IL-2; (c) rapidly expanding the second TIL population in a second cell culture medium using a myeloid artificial antigen presenting cell (myeloid aAPC) population to obtain a third TIL population; wherein the number of the third TIL population is 7 days after the start of the rapid amplification At least 50 times greater than the number of second TIL groups; wherein the second cell culture medium comprises IL-2 and OKT-3; (d) administering a therapeutically effective portion of the third TIL population to the cancer patient. In some embodiments, the method comprises a first step of: (a) the first TIL population was obtained from resected tumors of the patient. In some embodiments, in the second cell culture medium, IL-2 is present at an initial concentration of about 3000IU/mL and the OKT-3 antibody is present at an initial concentration of about 30 ng/mL. In some embodiments, the first amplification is performed for a period of no more than 14 days. In some embodiments, the first amplification is performed using a gas permeable container. In some embodiments, the second amplification is performed using a gas permeable container. In some embodiments, in rapid amplification, the ratio of the second TIL population to the aAPC population is 1: 80 to 1: 400. in some embodiments, in rapid amplification, the ratio of the second TIL population to the aAPC population is about 1: 300. in some embodiments, the cancer treated is selected from melanoma, ovarian cancer, cervical cancer, non-small cell lung cancer (NSCLC), lung cancer, bladder cancer, breast cancer, cancer caused by human papilloma virus, head and neck cancer including Head and Neck Squamous Cell Carcinoma (HNSCC), renal cancer, and renal epithelial renal cell carcinoma. In some embodiments, the cancer treated is selected from melanoma, ovarian cancer, and cervical cancer. In some embodiments, the cancer treated is melanoma. In some embodiments, the cancer treated is ovarian cancer. In some embodiments, the cancer treated is cervical cancer. In some embodiments, the method of treating cancer further comprises the step of treating the patient with a non-myeloablative lymphocyte depletion regimen prior to administering the third TIL population to the patient. In some embodiments, the non-myeloablative lymphocyte depletion protocol comprises the steps of: at 60mg/m ²Cyclophosphamide was administered for two days at a dose of 25mg/m²The dose of fludarabine was administered for five days. In some embodiments, the high dose IL-2 regimen comprises administering 600,000 or 720,000IU/kg aldesleukin, or a biological analog or variant thereof, as a 15 minute iv bolus every 8 hours until tolerated. In some embodiments, the TIL for treatment has been associated with one or more genes described herein (including PD-1, LAG-3, TIM-3, CIS)H and CBLB), the TIL for treatment may be added to the cell culture medium during the first amplification and/or the second amplification (e.g., according to step B, step C, and/or step D of fig. 8); wherein the TIL and other agents may be added in an amount selected from the group consisting of: mu.M sd-RNA/10,000 TIL/100. mu.L medium, 0.5. mu. Msd-RNA/10,000 TIL/100. mu.L medium, 0.75. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.25. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 1.5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 2. mu.M sd-RNA/10,000 TIL/100. mu.L medium, 5. mu.M sd-RNA/10,000 TIL/100. mu.L medium, or 10. mu.M sd-RNA/10,000 TIL/100. mu.L medium. In some embodiments, the TIL for treatment has been contacted with one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB); during the first amplification and/or the second amplification (e.g., according to steps B, C and/or D of fig. 8), one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB) can be added to the TIL culture twice a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. In one embodiment, the TIL for treatment has been contacted with one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB); wherein, during the first amplification and/or the second amplification (e.g. according to step B, step C and/or step D of fig. 8), TIL and other reagents may be added in an amount selected from the group consisting of: mu.M sd-RNA/10,000TIL, 0.5. mu.M sd-RNA/10,000TIL, 0.75. mu.M sd-RNA/10,000TIL, 1. mu.M sd-RNA/10,000TIL, 1.25. mu.M sd-RNA/10,000TIL, 1.5. mu.M sd-RNA/10,000TIL, 2. mu.M sd-RNA/10,000TIL, 5. mu.M sd-RNA/10,000TIL, or 10. mu.M sd-RNA/10,000 TIL. In one embodiment, the TIL for treatment has been contacted with one or more sd-RNAs targeting genes described herein (including PD-1, LAG-3, TIM-3, CISH, and CBLB); wherein the TIL and its nucleic acid sequence can be administered twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days during the first amplification and/or the second amplification (e.g., according to steps B, C and/or D of FIG. 8) He reagents were added to TIL cultures.

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Method for producing TIL products enriched with tumor antigen specific T cells

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