METHODS FOR IMPROVING THE EFFICACY AND EXPANSION OF CHIMERIC ANTIGEN RECEPTOR?EXPRESSING CELLS

• US 20180133296A1
• Filed: 04/15/2016
• Published: 05/17/2018
• Est. Priority Date: 04/17/2015
• Status: Active Grant

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Daniel J. Powell Jr
• Inventors: Barrett, David Maxwell, Bedoya, Felipe, Ghassemi, Saba, June, Carl H., Levine, Bruce L., Melenhorst, Jan J., Milone, Michael C., Powell, Jr., Daniel J., Singh, Nathan Amar, Zheng, Zoe
• Granted Patent: US 11,896,614 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/5156 expressing foreign proteins
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 2239/13 Antibody-based
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/59 Reproductive system, e.g. uterus, ovaries, cervix or testes
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• 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 38/50 acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
• 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 39/464412 CD19 or B4
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4748 Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70517 : CD8
• CPC Class Codes: C07K 14/70578 : NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95 NGF-receptor C07K14/71, TNF-receptor C07K14/7151
• CPC Class Codes: C07K 16/28 : against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2803 : against the immunoglobulin superfamily
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/03 : containing a transmembrane segment
• CPC Class Codes: C07K 2319/33 : fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C12N 2501/2302 : Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2305 : Interleukin-5 (IL-5)
• CPC Class Codes: C12N 2501/2307 : Interleukin-7 (IL-7)
• CPC Class Codes: C12N 2501/2318 : Interleukin-18 (IL-18)
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12Y 305/01001 : Asparaginase (3.5.1.1)

Abstract

The invention provides methods of making immune effector cells (e.g., T cells, NK cells) that can be engineered to express a chimeric antigen receptor (CAR), compositions and reaction mixtures comprising the same, and methods of treatment using the same.

Claims

1. A method of enriching for, or making, an immune effector cell (e.g., cell population) suitable for use in a CAR therapy, comprising acquiring (e.g., obtaining or harvesting) the immune effector cell, according to one, two, three, four, five or more (all) of the following:

(i) the timing of immune effector cell acquisition from a subject (e.g., a hematological cancer patient);

(ii) the timing of the chemotherapy in relation to the immune effector cell acquisition;

(iii) the type of chemotherapy;

(iv) the underlying malignancy;

(v) an immune effector cell (e.g., T cell) parameter, e.g., one or more of T cell number, T cell phenotype or T cell function,

or a combination of two, three, four, or five of (i)-(v),

thereby enriching for, or making, the immune effector cell suitable for use in a CAR therapy.

4. The method of any of claim 1-3, wherein the immune effector cell population shows an increase in one or more of:

ex-vivo expansion of the immune cell population, the efficacy of the immune cell population for therapy, or the yield of the immune cell population, when any of (i)-(v) are optimized.

8. The method of any of claims 1-7, wherein the subject from which immune cells are acquired and/or the subject to be treated, is a human cancer patient.

9. The method of claim 8, wherein the subject is 18 years of age of younger (e.g., 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or younger).

10. The method of claim 8, wherein the subject is older than 18 years of age.

11. The method of any of claims 1-10, wherein the subject has a disease associated with expression of a tumor- or cancer associated-antigen.

12. The method of any of claims 1-11, wherein the hematological cancer is chosen from one or more of:

a B-cell acute lymphocytic leukemia (B-ALL), T-cell acute lymphocytic leukemia (T-ALL), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B cell promyelocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt'"'"'s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin'"'"'s lymphoma (NHL), Hodgkin'"'"'s lymphoma (HL), plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, and Waldenstrom macroglobulinemia.

13. The method of any of claims 1-11, wherein the hematological cancer is a leukemia, e.g., CLL, ALL, or a lymphoma, e.g., MCL, NHL, or HL.

14. The method of any of claim 1-9 or 11-13, wherein the subject has a leukemia, and is a pediatric patient, e.g., is 18 years of age of younger.

15. The method of any of claims 1-14, wherein the subject is classified as having standard-risk, high-risk, or very high-risk ALL.

16. The method of any of claims 1-14, wherein the subject does not have a lymphoma, e.g., the subject does not have Non-Hodgkin lymphoma (NHL).

17. The method of any of claims 1-16, wherein the subject does not have a relapsed cancer.

18. The method of any of claims 1-17, wherein the immune effector cell population acquisition occurs prior to administration of the chemotherapy to the subject.

19. The method of any of claims 1-17, wherein the immune effector cell population acquisition occurs before the subject has undergone 2, 3, 4 or 5 cycles of chemotherapy.

20. The method of any of claims 1-17, wherein the immune effector cell population acquisition occurs after the subject has undergone 1 cycle of chemotherapy, but before the subject has undergone more than 1 cycle (e.g., more than 1, 2, 3, 4, or 5 cycles) of chemotherapy.

21. The method of any of claims 1-20, wherein the chemotherapy or cycle of chemotherapy comprises one or more of an induction, a consolidation, an interim maintenance, a delayed intensification, or a maintenance therapy cycle.

22. The method of claim 21, wherein the immune effector cell population is acquired from the subject before the subject has undergone a consolidation cycle of chemotherapy or an induction cycle of chemotherapy.

23. The method of claim 21, wherein the immune effector cell population is acquired from the subject before the subject has undergone a consolidation cycle of chemotherapy.

24. The method of claim 21, wherein the subject is classified as having a high-risk or very high-risk cancer (e.g., ALL) and the cells are harvested before the subject has undergone a consolidation cycle.

25. The method of claim 21, wherein the immune effector cell population is acquired from the subject before the subject has undergone a delayed intensification cycle.

26. The method of claim 21, wherein the subject is classified as having a high-risk or very high-risk cancer (e.g., ALL) and the cells are harvested before the subject has undergone a delayed intensification cycle.

27. The method of any of claims 1-26, wherein one or more chemotherapy cycles or drugs are chosen from Table 8.

28. The method of any of claims 1-27, wherein the chemotherapy comprises a drug and dosing regimen described in Table 8.

29. The method of any of claims 1-27, wherein the chemotherapy comprises one or more of vincristine, dexamethasone, PEG-L-asparaginase, daunorubicin, 6-mercaptopurine, cyclophosphamide, cytarabine, methotrexate, doxorubicin, 6-thioguanine, and/or prednisone.

30. The method of any of claims 1-27, wherein the immune effector cell population is acquired from the subject before the subject has been administered cyclophosphamide and/or cytarabine.

31. The method of any of claims 1-30, the acquired immune effector cell population comprises a higher number of less differentiated T cells, e.g., a higher number of one or more of naï

ve T cells, stem central memory T cells, and/or central memory T cells, e.g., compared to a reference value (e.g., a sample from the subject at a later time point or after exposure to additional rounds of chemotherapy).

33. The method of claim 31 or 32, wherein the acquired immune effector cell population includes at least 50% central memory T cells.

34. The method of claim 31 or 32, wherein the acquired immune effector cell population includes less than 55% effector memory and terminal effector T cells combined.

35. The method of claim 31, wherein the acquired immune effector cell population shows an increased absolute T cell count (ATC), compared to a reference value (e.g., a sample from the subject at a later time point or after exposure to additional rounds of chemotherapy).

36. The method of claim 31, wherein the acquired immune effector cell population comprises an absolute T cell count of at least 400 cells/microliter, an absolute naï

ve T cell count of at least 200 cells/microliter, an absolute stem central memory T cell count of at least 20 cells/microliter, and/or an absolute central memory T cell count of at least 40 cells/microliter.

37. The method of any of claims 1-36, wherein the acquired immune effector cell population is selected based upon the expression of one or more markers, e.g., CCR7, CD62L, CD45RO, and CD95, e.g., the population of immune effector cells (e.g., T cells) are CCR7+ and CD62L+.

39. The method of any of claims 1-38, wherein the immune cell population is acquired, from a subject having a haematological cancer, e.g., a leukemia, e.g., CLL, ALL, or a lymphoma, e.g., MCL, NHL, or HL.

40. The method of any of claims 1-39, further comprising removing T regulatory cells, e.g., CD25+ T cells, from the acquired immune cell population, to thereby provide a population of T regulatory-depleted cells, e.g., CD25+ depleted cells.

41. The method of claim 40, wherein the population of T regulatory-depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

42. The method of any of claims 1-41, wherein the acquired immune effector cell population are cells of a subject having cancer, e.g., a subject having a CD25-expressing cancer such as, e.g., chronic lymphocytic leukemia (CLL).

43. The method of claim 41, wherein the population of T regulatory-depleted cells contains less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of tumor cells.

44. The method of claim 40, wherein the T regulatory cells, e.g., CD25+ T cells, are removed from the acquired immune effector cell population using an anti-CD25 antibody, or fragment thereof.

45. The method of claim 44, wherein the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate, e.g., a bead.

46. The method of claim 43, wherein the population of T regulatory-depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of the leukemia cells, e.g., CLL cells, ALL cells, or lymphoma cells, e.g., MCL cells, HL cells.

47. The method of claim 43, wherein the population of T regulatory-depleted cells contains less than 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 15%, 10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CLL cells.

48. The method of claim 43, wherein the population of T regulatory-depleted cells contains less than 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells and less than 10%, 5%, 4%, 3%, 2%, 1% of tumor cells, e.g., CLL cells.

49. The method of any of claims 1-48, further comprising:

removing cells from the acquired immune effector cell population which express a tumor antigen, e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, to thereby provide a population of T regulatory-depleted, e.g., CD25+ depleted, and tumor antigen depleted cells that are suitable for expression of a CAR, e.g., a CAR described herein.

50. The method of any of claims 1-48, further comprising removing cells from the acquired immune effector cell population which express a check point inhibitor, e.g., a check point inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory-depleted, e.g., CD25+ depleted cells, and check point inhibitor depleted cells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells.

51. The method of any of claims 1-48, wherein the acquired immune effector cell population has been selected based upon the expression of one or more markers, e.g., CD3, CD28, CD4, CD8, CD45RA, and CD45RO, e.g., the provided population of immune effector cells (e.g., T cells) are CD3+ and/or CD28+.

52. The method of any of the preceding claims, further comprising:

activating the population of T regulatory-depleted cells, e.g., CD25+ depleted cells.

53. The method of any of the preceding claims, further comprising:

transducing a cell from the immune effector cell population with a vector comprising a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein.

54. The method of claim 53, further comprising expanding the immune effector cell population, e.g., engineered to express a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, e.g., by a method described herein.

55. The method of any of the preceding claims, further comprising:

transducing a cell from the population of T regulatory-depleted cells, e.g., the population of CD25+ depleted cells, with a vector comprising a nucleic acid encoding a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein.

56. The method of claim 55, further comprising expanding the population of T regulatory-depleted cells, e.g., engineered to express a CAR, e.g., a CAR described herein, e.g., a CD19 CAR described herein, e.g., by a method described herein.

57. The method of claim 54 or 56, wherein the population of cells is expanded for a period of 8 days or less, e.g., 7, 6 or 5 days.

58. The method of claim 54 or 56, wherein the population of cells is expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions.

59. The method of claim 58, wherein the cells expanded for 5 days show at least a one, two, three or four fold increase in cells doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions.

60. The method of claim 58, wherein the cells are expanded in culture for 5 days, and the resulting cells exhibit higher proinflammatory cytokine production, e.g., IFN-γ

and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions.

61. The method of claim 54 or 56, wherein the population of cells is expanded by culturing the cells in the presence of an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the cells, e.g., as described herein. In one embodiment, the agent is a bead conjugated with anti-CD3 antibody, or a fragment thereof, and/or anti-CD28 antibody, or a fragment thereof.

62. The method of any of claims 54-61-55, wherein the population of cells is expanded in an appropriate media (e.g., media described herein) that includes one or more interleukin that result in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in cells over a 14 day expansion period, e.g., as measured by a method described herein such as flow cytometry.

63. The method of claim 62, wherein the population of cells is expanded in the presence a cytokine, e.g., IL-15 and/or IL-7 (e.g., IL-15 and IL-7).

64. The method of any of claims 54-63, wherein the population of the cells is cryopreserved after the expansion period.

65. The method of any of the preceding claims further comprising contacting the population of immune effector cells with a nucleic acid encoding a telomerase subunit, e.g., hTERT.

2. A composition comprising an immune effector cell (e.g., cell population) that expresses a CAR molecule (a “

CAR-expressing cell”

) for use, in combination with a chemotherapy, in treating, or in providing anti-tumor immunity to, a subject having a hematological cancer, wherein the immune effector cell is acquired (e.g., obtained or harvested) from the subject, by optimizing one, two, three, four, or more (all) of the following;

(i) the timing of immune effector cell acquisition from a subject (e.g., a cancer patient);

(ii) the timing of the chemotherapy in relation to the immune effector cell acquisition;

(iii) the type of chemotherapy;

(iv) the underlying malignancy;

(v) an immune effector cell (e.g., T cell) parameter, e.g., one or more of T cell number, T cell phenotype or T cell function,

or a combination of two, three, four, or five of (i)-(v),

(optionally) wherein the immune effector cell is acquired from the subject prior to introduction of the CAR molecule,

5. The method of any of claims 2-4, wherein the CAR-expressing cell comprises a nucleic acid encoding a CAR, e.g., a CAR molecule described herein, e.g., a CD19 CAR described herein (e.g., CTL019).

6. The method of any of claims 2-5, further comprising introducing into the immune effector cell population a nucleic acid encoding a CAR, e.g., a CAR molecule described herein, e.g., a CD19 CAR described herein (e.g., CTL019).

7. The method of claim 5 or 6, wherein the CAR molecule comprises a sequence according to Table 1 or Table 4.

3. A method of treating, or providing anti-tumor immunity to, a subject having a hematological cancer, comprising administering to the subject an effective amount of an immune effector cell (e.g., a cell population) that expresses a CAR molecule (a “

CAR-expressing cell”

or a “

CAR therapy”

), in combination with a chemotherapy, wherein the immune effector cell is acquired (e.g., obtained or harvested) from the subject, by optimizing one, two, three, four, or more (all) of the following;

(i) the timing of immune effector cell acquisition from a subject (e.g., a cancer patient);

(ii) the timing of the chemotherapy in relation to the immune effector cell acquisition;

(iii) the type of chemotherapy;

(iv) the underlying malignancy;

(v) an immune effector cell (e.g., T cell) parameter, e.g., one or more of T cell number, T cell phenotype or T cell function,

or a combination of two, three, four, or five of (i)-(v),

(optionally) wherein the immune effector cell is acquired from the subject prior to introduction of the CAR molecule,

thereby treating, or providing anti-tumor immunity to, the hematological cancer.

32. The method of claim 231, wherein the acquired immune effector cell population includes at least 20% naï

ve T cells, at least 2% stem central memory T cells, and/or at least 4% central memory T cells.

38. The method of claim 32, wherein the naï

ve T cells are identified based upon an expression pattern of CCR7+, CD62L+, CD45RO−

, CD95−

, wherein the stem central memory T cells are identified based upon an expression pattern of CCR7+, CD62L+, CD45RO−

, CD95+, and wherein the central memory T cells are identified based upon an expression pattern of CCR7+, CD62L+, CD45RO+, CD95+.

66. An immune cell preparation or reaction mixture, e.g., comprising a population of immune effector cells (e.g., comprising a CAR molecule or a nucleic acid encoding a CAR molecule), made according to any of the methods described herein.

67. The immune effector cell preparation (e.g., reaction mixture) of claim 66, which is chosen from:

(i) the population of immune effector cells comprises at least 20% naï

ve T cells, at least 2% stem central memory T cells, and/or at least 4% central memory T cells, and/or (ii) the population of immune effector cells comprises an absolute T cell count of at least 400 cells/microliter, an absolute naï

ve T cell count of at least 200 cells/microliter, an absolute stem central memory T cell count of at least 20 cells/microliter, and/or an absolute central memory T cell count of at least 40 cells/microliter.

68. The immune effector cell preparation (e.g., reaction mixture) of claim 66, which has been selected based upon the expression of one or more markers, e.g., CCR7, CD62L, CD45RO, and CD95, e.g., the population of immune effector cells (e.g., T cells) are CCR7+ and CD62L+.

69. The immune effector cell preparation (e.g., reaction mixture) of claim 67, wherein the naï

ve T cells are identified based upon an expression pattern of CCR7+, CD62L+, CD45RO−

, CD95−

, wherein the stem central memory T cells are identified based upon an expression pattern of CCR7+, CD62L+, CD45RO−

, CD95+, and wherein the central memory T cells are identified based upon an expression pattern of CCR7+, CD62L+, CD45RO+, CD95+.

70. The immune effector cell preparation (e.g., reaction mixture) of any of claims 67-69, which comprises a nucleic acid encoding a CAR, e.g., a CAR as described herein.