IMPROVED T CELL COMPOSITIONS

• US 20170218337A1
• Filed: 06/05/2015
• Published: 08/03/2017
• Est. Priority Date: 06/06/2014
• Status: Active Grant

• Current Assignee: 2seventy Bio, Inc. (bluebird bio, Inc.)
• Original Assignee: bluebird bio, Inc.
• Inventors: Friedman, Kevin
• Granted Patent: US 10,479,975 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/46 characterised by the cancer treated
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/4631 Chimeric Antigen Receptors [CAR]
• CPC Class Codes: A61K 39/464417 Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
• CPC Class Codes: A61P 1/18 for pancreatic disorders, e.g. pancreatic enzymes
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/10 of the bladder
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/74 containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C12N 2501/2302 : Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/51 : B7 molecules, e.g. CD80, CD86, CD28 (ligand), CD152 (ligand)
• CPC Class Codes: C12N 2501/515 : CD3, T-cell receptor complex
• CPC Class Codes: C12N 2501/599 : with CD designations not provided for elsewhere
• CPC Class Codes: C12N 2501/727 : Kinases (EC 2.7.)
• CPC Class Codes: C12N 2501/999 : Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2510/00 : Genetically modified cells
• CPC Class Codes: C12N 5/0636 : T lymphocytes

Abstract

The invention provides improved T cell compositions and methods for manufacturing T cells. More particularly, the invention provides methods of T cell manufacturing that result in adoptive T cell immunotherapies with improved survival, expansion, and persistence in vivo.

Claims

1. A method for manufacturing T cells comprising:

(a) activating a population of T cells and stimulating the population of T cells to proliferate, wherein the activation and stimulation steps are performed in the presence of an inhibitor of AKT/mTOR pathway;

(b) transducing the T cells with a viral vector comprising an engineered T cell receptor (TCR) or a chimeric antigen receptor (CAR);

(c) culturing the transduced T cells to proliferate;

wherein the activating and stimulating steps performed in the presence of the inhibitor of the PI3K/AKT/mTOR pathway results in maintaining proliferation of the transduced T cells compared to the proliferation of transduced T cells that were activated and stimulated in the absence of the inhibitor of the PI3K/AKT/mTOR pathway.

2. The method of claim 1, wherein the method comprises isolating peripheral blood mononuclear cells as the source of T cells.

3. The method of claim 1, wherein activation of the T cells comprises contacting the T cells with an anti-CD3 antibody or CD3-binding fragment thereof.

4. The method of claim 1, wherein stimulation of the T cells comprises contacting the T cells with an anti-CD28 antibody or a CD28-binding fragment thereof, B7-1 or a CD28-binding fragment thereof, or B7-2 or a CD28-binding fragment thereof.

5. The method of claim 1, wherein the cells are transduced with the viral vector prior to T cell proliferation.

6. The method of claim 1, wherein the cells are transduced with the viral vector after T cell proliferation.

7. The method of any one of claims 1 to 6, wherein the vector is a retroviral vector.

8. The method of any one of claims 1 to 7, wherein the vector is a lentiviral vector.

9. The method of any one of claims 1 to 8, wherein the cells comprise a chimeric antigen receptor (CAR).

10. The method of claim 9, wherein the CAR comprises

a) an extracellular domain that binds an antigen selected from the group consisting of:

alpha folate receptor, 5T4, α

_vβ

₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα

, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα

, IL-13Rα

2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, and VEGFR2;

b) a transmembrane domain derived from a polypeptide selected from the group consisting of;

CD8α

;

CD4, CD28, CD45, PD-1, and CD152;

c) one or more intracellular costimulatory signaling domains selected from the group consisting of;

CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), and CD278 (ICOS); and

d) a CD3ζ

signaling domain.

11. The method of claim 10, wherein the extracellular domain comprises an antibody or antigen binding fragment that binds the antigen.

12. The method of claim 10, wherein the transmembrane domain is derived from CD8α

or CD28.

13. The method of claim 10, wherein the one or more costimulatory signaling domains selected from the group consisting of:

CD28, CD134, and CD137.

14. The method of claim 10, further comprising a hinge region polypeptide.

15. The method of claim 14, wherein the hinge region polypeptide comprises a hinge region of IgG1 or CD8α

.

16. The method of claim 10, further comprising a signal peptide.

17. The method of claim 16, wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α

signal polypeptide.

18. The method of any one of claims 1 to 17, wherein the inhibitor of the PI3K/AKT/mTOR pathway is selected from the group consisting of:

BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121.

19. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a pan-PI3K inhibitor selected from the group consisting of:

BEZ235, LY294002, and GDC-0941.

20. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a selective PI3K inhibitor selected from the group consisting of:

BYL719, GSK2636771, TGX-221, AS25242, CAL-101, and IPI-145.

21. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is the PI3-K inhibitor ZSTK474.

22. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a pan-AKT inhibitor selected from the group consisting of:

MK-2206, GSK690693, and GDC-0068.

23. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is the selective AKT1 inhibitor A-674563.

24. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is the selective AKT2 inhibitor CCT128930.

25. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits DNA-PK activation of AKT.

26. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits PDK-1 activation of AKT.

27. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits mTORC2 activation of AKT.

28. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits HSP activation of AKT.

29. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a pan-mTOR inhibitor selected from the group consisting of:

AZD8055, INK128, and rapamycin.

30. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a selective mTORC1 inhibitor selected from the group consisting of:

PF-04691502 and everolimus.

31. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a s6 kinase inhibitor selected from the group consisting of:

BI-D1870, H89, PF-4708671, FMK, and AT7867.

32. The method of claim 18, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a DNA-PK inhibitor selected from the group consisting of:

NU7441, PI-103, NU7026, PIK-75, and PP-121.

33. The method of any one of claims 1 to 32, wherein the population of T cells activated and stimulated in the presence of an inhibitor of PI3K/AKT/mTOR pathway have an increased number of T cells expressing one or more markers selected from the group consisting of:

CD62L, CCR7, CD28, CD27, CD122, and CD127 compared to a population of T cells activated and stimulated in the absence of the inhibitor of PI3K/AKT/mTOR pathway.

34. The method of any one of claim 33, wherein the population of T cells activated and stimulated in the presence of an inhibitor of PI3K/AKT/mTOR pathway do not express CD57 or KLRG1 or express less CD57 or KLRG1 compared to a population of T cells activated and stimulated in the absence of the inhibitor of PI3K/AKT/mTOR pathway.

35. A method for maintaining the proliferation and decreasing the differentiation of restimulated T cells expressing an engineered TCR or CAR comprising:

(a) contacting all or a portion of a population of proliferated T cells comprising an engineered TCR or CAR with an anti-CD3 antibody or CD3-binding fragment thereof, and an anti-CD28 antibody or CD28-binding fragment thereof, which stimulates a CD28 accessory molecule on the surface of the T cells, thereby restimulating the activated T cells to proliferate;

wherein the restimulated T cells have maintained proliferation and decreased differentiation compared to the proliferation of T cells that were stimulated or restimulated in the absence of the inhibitor of the PI3K/AKT/mTOR pathway.

36. The method of claim 35, wherein the cells comprise an engineered TCR.

37. The method of claim 35, wherein the cells comprise a CAR.

38. The method of any one of claims 35 to 37, wherein the cells comprise a viral vector encoding an engineered TCR or CAR.

39. The method of claim 38, wherein the vector is a retroviral vector.

40. The method of claim 39, wherein the vector is a lentiviral vector.

41. The method of an one of claims 37 to 40, wherein the CAR comprises

a) an extracellular domain that binds an antigen selected from the group consisting of:

alpha folate receptor, 5T4, α

_vβ

₆ integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα

, GD2, GD3, ′

Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα

, IL-13Rα

2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, and VEGFR2;

b) a transmembrane domain derived from a polypeptide selected from the group consisting of;

CD8α

;

CD4, CD28, CD45, PD-1, and CD152;

c) one or more intracellular costimulatory signaling domains selected from the group consisting of;

CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), and CD278 (ICOS); and

d) a CD3ζ

signaling domain.

42. The method of claim 41, wherein the extracellular domain comprises an antibody or antigen binding fragment that binds the antigen.

43. The method of claim 41, wherein the transmembrane domain is derived from CD8α

or CD28.

44. The method of claim 41, wherein the one or more costimulatory signaling domains selected from the group consisting of:

CD28, CD134, and CD137.

45. The method of claim 41, further comprising a hinge region polypeptide.

46. The method of claim 45, wherein the hinge region polypeptide comprises a hinge region of IgG1 or CD8α

.

47. The method of claim 41, further comprising a signal peptide.

48. The method of claim 47, wherein the signal peptide comprises an IgG1 heavy chain signal polypeptide or a CD8α

signal polypeptide.

49. The method of any one of claims 35 to 48, wherein the inhibitor of the PI3K/AKT/mTOR pathway is selected from the group consisting of:

BEZ235, LY294002, GDC-0941, BYL719, GSK2636771, TGX-221, AS25242, CAL-101, IPI-145, MK-2206, GSK690693, GDC-0068, A-674563, CCT128930, AZD8055, INK128, rapamycin, PF-04691502, everolimus, BI-D1870, H89, PF-4708671, FMK, AT7867, NU7441, PI-103, NU7026, PIK-75, ZSTK474, and PP-121.

50. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a pan-PI3K inhibitor selected from the group consisting of:

BEZ235, LY294002, and GDC-0941.

51. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a selective PI3K inhibitor selected from the group consisting of:

BYL719, GSK2636771, TGX-221, AS25242, CAL-101, and IPI-145.

52. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is the PI3-K inhibitor ZSTK474.

53. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a pan-AKT inhibitor selected from the group consisting of:

MK-2206, GSK690693, and GDC-0068.

54. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is the selective AKT1 inhibitor A-674563.

55. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is the selective AKT2 inhibitor CCT128930.

56. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits DNA-PK activation of AKT.

57. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits PDK-1 activation of AKT.

58. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits mTORC2 activation of AKT.

59. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway inhibits HSP activation of AKT.

60. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a pan-mTOR inhibitor selected from the group consisting of:

AZD8055, INK128, and rapamycin.

61. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a selective mTORC1 inhibitor selected from the group consisting of:

PF-04691502 and everolimus.

62. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a s6 kinase inhibitor selected from the group consisting of:

BI-D1870, H89, PF-4708671, FMK, and AT7867.

63. The method of claim 49, wherein the inhibitor of the PI3K/AKT/mTOR pathway is a DNA-PK inhibitor selected from the group consisting of:

NU7441, PI-103, NU7026, PIK-75, and PP-121.

64. The method of any one of claims 35 to 63, wherein the population of activated T cells restimulated in the presence of an inhibitor of PI3K/AKT/mTOR pathway have an increased number of T cells expressing one or more markers selected from the group consisting of:

CD62L, CCR7, CD28, CD27, CD122, and CD127 compared to a population of T cells activated and stimulated in the absence of the inhibitor of PI3K/AKT/mTOR pathway.

65. The method of any one of claim 64, wherein the population of activated T cells restimulated in the presence of an inhibitor of PI3K/AKT/mTOR pathway do not express CD57 or KLRG1 or express less CD57 or KLRG1 compared to a population of T cells activated and stimulated in the absence of the inhibitor of PI3K/AKT/mTOR pathway.

66. A population of T cells comprising a vector comprising an engineered TCR or CAR, wherein the cells have been activated and stimulated to proliferate in the presence of an inhibitor of PI3K/AKT/mTOR pathway.

68. The population of immune effector cells of claim 66 or claim 67, wherein the immune effector cells comprise T cells.

69. A composition comprising the population of immune effector cells of claim 66 or 67 and a physiologically acceptable excipient.

70. A method of treating a cancer in a subject in need thereof, comprising administering to the subject a therapeutically effect amount of the composition of claim 69.

71. The method of claim 70, wherein the cancer is selected from the group consisting of Wilms'"'"' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin'"'"'s lymphoma, non-Hodgkin'"'"'s lymphoma, and urinary bladder cancer.

72. The method of claim 70, wherein the cancer is pancreatic cancer and the extracellular binding domain binds an epitope of PSCA or MUC1

73. The method of claim 70, wherein the cancer is bladder cancer and the extracellular binding domain binds an epitope of PSCA or MUC1

74. The method of claim 70, wherein the cancer is glioblastoma multiforme and the extracellular binding domain binds an epitope of EPHA2, EGFRvIII, or CSPG4.

75. The method of claim 70, wherein the cancer is lung cancer and the extracellular binding domain binds an epitope of PSCA or GD2.

76. The method of claim 70, wherein the cancer is breast cancer and the extracellular binding domain binds an epitope of CSPG4 or HER2.

77. The method of claim 70, wherein the cancer is melanoma and the extracellular binding domain binds an epitope of CSPG4 or GD2.

78. The method of claim 70, wherein the cancer is a B-cell malignancy and the binding domain binds an epitope of BCMA.

79. A method of treating a hematological malignancy in a subject in need thereof, comprising administering to the subject a therapeutically effect amount of the composition of claim 69.

80. The method of claim 79, wherein the hematological malignancy is a B-cell malignancy selected from the group consisting of:

multiple myeloma (MM), chronic lymphocytic leukemia (CLL), or non-Hodgkin'"'"'s lymphoma (NHL).

81. The method of claim 80, wherein the MM is selected from the group consisting of:

overt multiple myeloma, smoldering multiple myeloma, plasma cell leukemia, non-secretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary plasmacytoma of bone, and extramedullary plasmacytoma.

82. The method of claim 80, wherein the NHL is selected from the group consisting of:

Burkitt lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma.

67. A population of T cells comprising a vector comprising an engineered TCR or CAR, wherein the cells have been activated and stimulated to proliferate in the presence of an inhibitor of PI3K/AKT/mTOR pathway and have been restimulated by contacting all or a portion of a population of proliferated immune effector cells with an anti-CD3 antibody or CD3-binding fragment thereof, and an anti-CD28 antibody or CD28-binding fragment thereof, which stimulates a CD28 accessory molecule on the surface of the immune effector cells.