CAR T CELL THERAPIES WITH ENHANCED EFFICACY

• US 20180258149A1
• Filed: 09/16/2016
• Published: 09/13/2018
• Est. Priority Date: 09/17/2015
• Status: Abandoned Application

• Current Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Original Assignee: Novartis Ag, Trustees Of The University Of Pennsylvania (University of Pennsylvania)
• Inventors: Motz, Gregory, Bushman, Frederic Dixon, Fraietta, Joseph A., June, Carl H., Melenhorst, Jan J., Nobles, Christopher Loren, Young, Regina M.
• Application Number:
  US15/760,519
• Publication Number:
  US 20180258149A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 2039/5154 Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
• 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 2239/48 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 39/0011 Cancer antigens
• CPC Class Codes: A61K 39/001104 Epidermal growth factor receptors [EGFR]
• CPC Class Codes: A61K 39/001106 Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
• CPC Class Codes: A61K 39/001108 Platelet-derived growth factor receptors [PDGFR]
• CPC Class Codes: A61K 39/001109 Vascular endothelial growth factor receptors [VEGFR]
• CPC Class Codes: A61K 39/001112 CD19 or B4
• CPC Class Codes: A61K 39/001113 CD22, BL-CAM, siglec-2 or sialic acid- binding Ig-related lectin 2
• CPC Class Codes: A61K 39/001119 Receptors for interleukins [IL]
• CPC Class Codes: A61K 39/001122 Ephrin Receptors [Eph]
• CPC Class Codes: A61K 39/001124 CD20
• CPC Class Codes: A61K 39/001126 CD38 not IgG
• CPC Class Codes: A61K 39/001129 Molecules with a "CD" designation not provided for elsewhere
• CPC Class Codes: A61K 39/001149 Cell cycle regulated proteins, e.g. cyclin, CDC, CDK or INK-CCR
• CPC Class Codes: A61K 39/00115 Apoptosis related proteins, e.g. survivin or livin
• CPC Class Codes: A61K 39/001151 p53
• CPC Class Codes: A61K 39/001153 : Wilms tumor 1 [WT1]
• CPC Class Codes: A61K 39/001156 : Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]
• CPC Class Codes: A61K 39/001157 : Telomerase or TERT [telomerase reverse transcriptase]
• CPC Class Codes: A61K 39/001164 : GTPases, e.g. Ras or Rho
• CPC Class Codes: A61K 39/001166 : Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
• CPC Class Codes: A61K 39/001168 : Mesothelin [MSLN]
• CPC Class Codes: A61K 39/00117 : Mucins, e.g. MUC-1
• CPC Class Codes: A61K 39/001171 : Gangliosides, e.g. GM2, GD2 or GD3
• CPC Class Codes: A61K 39/001176 : Heat shock proteins
• CPC Class Codes: A61K 39/001182 : Carcinoembryonic antigen [CEA]
• CPC Class Codes: A61K 39/001186 : MAGE
• CPC Class Codes: A61K 39/001188 : NY-ESO
• CPC Class Codes: A61K 39/001191 : Melan-A/MART
• CPC Class Codes: A61K 39/001192 : Glycoprotein 100 [Gp100]
• CPC Class Codes: A61K 39/001193 : Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
• CPC Class Codes: A61K 39/001195 : Prostate specific membrane antigen [PSMA]
• CPC Class Codes: A61K 39/001197 : Breakpoint cluster region-abelson tyrosine kinase [BCR-ABL]
• CPC Class Codes: A61K 39/39558 : against tumor tissues, cells, antigens
• 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 39/464417 : Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: C07K 14/4748 : Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
• CPC Class Codes: C07K 14/70503 : Immunoglobulin superfamily
• CPC Class Codes: C12N 5/0636 : T lymphocytes
• CPC Class Codes: C12N 9/0069 : acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Y 113/11 : with incorporation of two atoms of oxygen (1.13.11)

Abstract

The invention provides compositions and methods improved CAR T cell therapies. Specifically, the invention provides cells with reduced Tet, e.g., Tet2 function or expression, and methods of use therefore. The invention further provides Tet2 inhibitors and methods of use therefore in connection with CAR T cells.

Claims

1. A cell (e.g., a population of cells) engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein expression and/or function of Tet1, Tet2 and/or Tet3 in said cell has been reduced or eliminated.

2. The cell of claim 1, wherein the antigen-binding domain binds to a tumor antigen is selected from a group consisting of:

TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

3. The cell of claim 2, wherein the tumor antigen is CD19.

4. The cell of claim 1, wherein the antigen-binding domain is an antibody or antibody fragment as described in, e.g., WO2012/079000 or WO2014/153270.

5. The cell of any of the preceding claims, wherein the transmembrane domain comprises:

an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO;

12, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

12;

or

the sequence of SEQ ID NO;

12.

6. The cell of any of the preceding claims, wherein the antigen binding domain is connected to the transmembrane domain by a hinge region, wherein said hinge region comprises SEQ ID NO:

2 or SEQ ID NO;

6, or a sequence with 95-99% identity thereof.

7. The cell of any of the preceding claims, wherein the intracellular signaling domain comprises a primary signaling domain and/or a costimulatory signaling domain, wherein the primary signaling domain comprises a functional signaling domain of a protein chosen from CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fcgamma RIIa, DAP10, or DAP12.

8. The cell of any of the preceding claims, wherein the primary signaling domain comprises:

an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO;

18 or SEQ ID NO;

20, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

18 or SEQ ID NO;

20;

or

the amino acid sequence of SEQ ID NO;

18 or SEQ ID NO;

20.

9. The cell of any of the preceding claims, wherein the intracellular signaling domain comprises a costimulatory signaling domain, or a primary signaling domain and a costimulatory signaling domain, wherein the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D.

10. The cell of any of the preceding claims, wherein the costimulatory signaling domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:

14 or SEQ ID NO;

16, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO;

14 or SEQ ID NO;

16.

11. The cell of any of the preceding claims, wherein the costimulatory signaling domain comprises a sequence of SEQ ID NO:

14 or SEQ ID NO;

16.

12. The cell of any of the preceding claims, wherein the intracellular domain comprises the sequence of SEQ ID NO:

14 or SEQ ID NO;

16, and the sequence of SEQ ID NO;

18 or SEQ ID NO;

20, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

13. The cell of any of the preceding claims, further comprising a leader sequence comprises the sequence of SEQ ID NO:

2.

14. The cell of any of the preceding claims, wherein the cell is an immune effector cell (e.g., a population of immune effector cells).

15. The cell of claim 14, wherein the immune effector cell is a T cell or an NK cell.

16. The cell of claim 15, wherein the immune effector cell is a T cell.

17. The cell of claim 16, wherein the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof.

18. The cell of any of the preceding claims, wherein the cell is a human cell.

19. The cell of any of the preceding claims, wherein the cell comprises an inhibitor of Tet1, Tet2, and/or Tet3.

20. The cell claim 19, wherein the inhibitor of Tet1, Tet2 and/or Tet3 is (1) a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3 or its regulatory elements, e.g., Tet2, or its regulatory elements;

(2) nucleic acid encoding one or more components of said gene editing system;

or (3) combinations thereof.

21. The cell of claim 20, wherein the gene editing system is selected from the group consisting of:

a CRISPR/Cas9 system, a zinc finger nuclease system, a TALEN system and a meganuclease system.

22. The cell of claim 20 or 21, wherein the gene editing system binds to a target sequence in an early exon or intron of a gene encoding Tet1, Tet2 and/or Tet3, e.g., Tet2.

23. The cell of claim 22, wherein the gene editing system binds a target sequence of a gene encoding tet2, and the target sequence is upstream of exon 4, e.g., in exon1, exon2, or exon3, e.g. in exon 3.

24. The cell of any of claims 20-23, wherein the gene editing system binds to a target sequence in a late exon or intron of a gene encoding Tet1, Tet2 and/or Tet3, e.g., Tet2.

25. The cell of claim 24, wherein the gene editing system binds a target sequence of a gene encoding tet2, and the target sequence is downstream of exon 8, e.g., is in exon9, exon10, or exon11, e.g. is in exon 9.

26. The cell of any of claims 18-25, wherein the gene editing system is a CRISPR/Cas system comprising a gRNA molecule comprising a targeting sequence which hybridizes to a target sequence of a Tet2 gene.

27. The cell of claim 26, wherein the targeting sequence is a targeting sequence listed in Table 3.

28. The cell of claim 26, wherein the targeting sequence is a targeting sequence listed in Table 5.

29. The cell of claim 19, wherein the inhibitor of Tet2 is an siRNA or shRNA specific for Tet1, Tet2, Tet3, or nucleic acid encoding said siRNA or shRNA.

30. The cell of claim 29, wherein the siRNA or shRNA comprises a sequence complementary to a sequence of a Tet2 mRNA, e.g., comprises a target sequence of shRNA listed in Table 4.

31. The cell of claim 19, wherein the inhibitor of Tet1, Tet2 and/or Tet3 is a small molecule.

32. The cell of claim 19, wherein the inhibitor of Tet1, Tet2, and/or Tet3 is a protein, e.g., is a dominant negative binding partner of Tet1, Tet2, and/or Tet3 (e.g., a histone deacetylase (HDAC) that interacts with Tet1, Tet2, and/or Tet3), or nucleic acid encoding said dominant negative binding partner of Tet1, Tet2, and Tet3.

33. The cell of claim 19, wherein the inhibitor of Tet1, Tet2, and/or Tet3 is a protein, e.g., is a dominant negative (e.g., catalytically inactive) Tet1, Tet2, or Tet3, or nucleic acid encoding said dominant negative Tet1, Tet2, or Tet3.

34. A method of increasing the therapeutic efficacy of a CAR-expressing cell, e.g., a cell of any of the preceding claims, e.g., a CAR19-expressing cell (e.g., CTL019), comprising a step of decreasing the level of 5-hydroxymethylcytosine in said cell.

35. A method of increasing the therapeutic efficacy of a CAR-expressing cell, e.g., a cell of any of the preceding claims, e.g., a CAR19-expressing cell (e.g., CTL019), comprising a step of contacting said cell with Tet inhibitor, e.g., a Tet1, Tet2 and/or Tet3 inhibitor.

36. The method of claim 34, wherein said step comprises contacting said cells with a Tet inhibitor.

37. The method of claim 35 or 36, wherein said Tet inhibitor is a Tet2 inhibitor.

38. The method of claim 36, wherein the Tet inhibitor is selected from the group consisting of:

(1) a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2, or Tet3, or its corresponding regulatory elements;

(2) a nucleic acid (e.g., an siRNA or shRNA) that inhibits expression of Tet1, Tet2, or Tet3;

(3) a protein (e.g., a dominant negative, e.g., catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner of Tet1, Tet2, or Tet3;

(4) a small molecule that inhibits expression and/or function of Tet1, Tet2, or Tet3;

(5) a nucleic acid encoding any of (1)-(3); and

(6) any combination of (1)-(5).

39. The method of claim 38, wherein the Tet inhibitor is a Tet2 inhibitor.

40. The method of any of claims 36-39, wherein said contacting occurs ex vivo.

41. The method of any of claims 36-39, wherein the contacting occurs in vivo.

42. The method of claim 41, wherein the contacting occurs in vivo prior to delivery of nucleic acid encoding a CAR into the cell.

43. The method of claim 41, wherein the contacting occurs in vivo after the cells have been administered to a subject in need thereof.

44. A cell for use in a method of treating a subject in need thereof, the method comprising administering to said subject an effective amount of the cell of any of claims 1-33.

45. The cell for use of claim 44, wherein the method further comprises administering to said subject a Tet1, Tet2, and/or Tet3 inhibitor.

50. The CAR-expressing cell therapy for use of any of claims 44-49, wherein the subject has a disease associated with expression of a tumor antigen, e.g., a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the tumor antigen.

51. The CAR-expressing cell therapy for use of claim 50, wherein the cancer is a hematologic cancer chosen from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic 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, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin'"'"'s lymphoma, Hodgkin'"'"'s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

52. The CAR-expressing cell therapy for use of claim 50, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'"'"'s Disease, non-Hodgkin'"'"'s lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi'"'"'s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

56. The method of any of claims 40-55, wherein the inhibitor is a Tet2 inhibitor.

69. A population of cells comprising one or more cells of any of claims 1-33, wherein the population of cells comprises a higher percentage of Tscm cells (e.g., CD45RA+CD62L+CCR7+CD27+CD95+ T cells) than a population of cells which does not comprise one or more cells in which expression and/or function of Tet1, Tet2 and/or Tet3 in said cell has been reduced or eliminated.

46. A CAR-expressing cell therapy for use in a method of treating a subject in need thereof, the method comprising administering to said subject the CAR-expressing cell therapy and a Tet1, Tet2, and/or Tet3 inhibitor.

47. The CAR-expressing cell therapy for use of claim 46, wherein the subject receives a pre-treatment of the Tet1, Tet2 and/or Tet3 inhibitor, prior to the initiation of the CAR-expressing cell therapy.

48. The CAR-expressing cell therapy for use of claim 46, wherein the subject receives concurrent treatment with a Tet1, Tet2, and/or Tet3 inhibitor and the CAR expressing cell therapy.

49. The CAR-expressing cell therapy for use of claim 46, wherein the subject receives treatment with a Tet1, Tet2, and/or Tet3 inhibitor post-CAR-expressing cell therapy.

53. A Tet1, Tet2 and/or Tet3 inhibitor, for use in the treatment of a subject, wherein said subject has received, is receiving, or is about to receive therapy comprising a CAR-expressing cell.

54. A method of manufacturing a CAR-expressing cell, comprising introducing nucleic acid encoding a CAR into a cell such that said nucleic acid (or CAR-encoding portion thereof) integrates into the genome of the cell within a Tet1, Tet2 and/or Tet3 gene (e.g., within an intron or exon of a Tet1, Tet2 and/or Tet3 gene), such that Tet1, Tet2 and/or Tet3 expression and/or function is reduced or eliminated.

55. A method of manufacturing a CAR-expressing cell, comprising contacting said CAR-expressing cell ex vivo with a Tet1, Tet2 and/or Tet3 inhibitor.

57. A vector comprising sequence encoding a CAR and sequence encoding a Tet inhibitor, e.g., a Tet1, Tet2, and/or Tet3 inhibitor.

58. The vector of claim 57, wherein the Tet inhibitor is a (1) a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2, or Tet3, or its corresponding regulatory elements;

(2) a nucleic acid (e.g., an siRNA or shRNA) that inhibits expression of Tet1, Tet2, or Tet3;

(3) a protein (e.g., a dominant negative, e.g., catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner of Tet1, Tet2, or Tet3; and

(4) a nucleic acid encoding any of (1)-(3), or combinations thereof.

59. The vector of claim 57 or 58, wherein the sequence encoding a CAR and the sequence encoding a Tet inhibitor are separated by a 2A site.

60. A gene editing system that is specific for a sequence of a Tet gene or its regulatory elements, e.g., a Tet1, Tet2 or Tet3 gene or its regulatory elements.

62. The gene editing system of claim 60 or 61, wherein the gene editing system is (1) a CRISPR/Cas gene editing system, (2) a zinc finger nuclease system, a TALEN system and a meganuclease system.

63. The gene editing system of claim 62, wherein the gene editing system is a CRISPR/Cas gene editing system.

64. The gene editing system of claim 63, comprising:

a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a Cas9 protein;

a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a nucleic acid encoding a Cas9 protein;

a nucleic acid encoding a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a Cas9 protein;

or

a nucleic acid encoding a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a nucleic acid encoding a Cas9 protein.

65. The gene editing system of any of claims 60-64, further comprising a template DNA.

66. The gene editing system of claim 65, wherein the template DNA comprises nucleic acid sequence encoding a CAR, e.g., a CAR as described herein.

61. The gene editing system of claim 61, wherein the gene editing system is specific for a sequence of a Tet2 gene.

67. A composition for the ex vivo manufacture of a CAR-expressing cell, comprising a Tet inhibitor, e.g., a Tet1, Tet2, and/or Tet3 inhibitor, e.g., a Tet2 inhibitor.

68. The composition of claim 67, wherein the Tet inhibitor is selected from N-[3-[7-(2,5-dimethyl-2H-pyrazol-3-ylamino)-1-methyl-2-oxo-1,4-dihydro-2H-pyrimido[4,5-d]pyrimidin-3-yl]-4-methylphenyl]-3-trifluoromethyl-benzamide, 2-[(2,6-dichloro-3-methylphenyl)amino]benzoic acid and 2-hydroxyglutarate.