MUC16 specific chimeric antigen receptors and uses thereof

1. A method of stimulating the proliferation and/or survival of engineered cells, the method comprising transfecting a cell obtained from a subject with a vector encoding a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and a cytoplasmic domain, to provide a genetically-engineered cell expressing the chimeric antigen receptor, wherein the antigen-binding domain comprises: (i) the amino acid sequence of SEQ ID NO: 5; and (ii) the amino acid sequence of SEQ ID NO: 6.

2. The method of claim 1 , wherein the vector further encodes a cytokine.

3. The method of claim 2 , wherein the vector encodes a fusion protein comprising: IL-15, or a functional fragment of variant thereof; and IL-15Rα, or a functional fragment or variant thereof.

4. The method of claim 3 , wherein the fusion protein comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 69.

5. The method of claim 2 , wherein the cytokine is IL 1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL18, IL-19, IL-20, TL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, TL-31, IL-32, TL-33, or a functional fragment or variant of any of the foregoing.

6. The method of claim 2 , wherein the cytokine is IL-15 or a functional fragment or variant thereof.

7. The method of claim 1 , wherein the vector is a lentivirus vector, a retroviral vector, or a non-viral vector.

8. The method of claim 7 , wherein the non-viral vector is a Tc1/mariner-type transposon.

9. The method of claim 7 , wherein the vector is a non-viral vector.

10. The method of claim 1 , further comprising transfecting the cell with a vector encoding a transposase.

11. The method of claim 10 , wherein the transposase is a Tc1/mariner-type transposase.

12. The method of claim 11 , wherein the Tc1/mariner-type transposase is SB 11, SB100X or SB110.

13. The method of claim 11 , wherein the Tc1/mariner-type transposase is SB 11.

14. The method of claim 1 , wherein the transmembrane domain comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 20.

15. The method of claim 1 , wherein the cytoplasmic domain comprises a cytoplasmic signaling sequence derived from CD3 zeta.

16. The method of claim 15 , wherein the cytoplasmic domain comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 26.

17. The method of claim 1 , wherein the cytoplasmic domain comprises a 4-1BB and/or a CD28 costimulatory signaling domain.

18. The method of claim 17 , wherein the cytoplasmic domain comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 22.

19. The method of claim 17 , wherein the cytoplasmic domain comprises: (a) a CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-13, DAP10, and/or DAP12 costimulatory signaling domain; and/or (b) a ligand that specifically binds with CD83.

20. The method of claim 17 , wherein the cytoplasmic domain comprises a 4-11BB and a CD28 costimulatory signaling domain.

21. The method of claim 17 , wherein the cytoplasmic domain comprises a 4-1BB costimulatory signaling domain.

22. The method of claim 17 , wherein the cytoplasmic domain comprises a CD28 costimulatory signaling domain.

23. The method of claim 1 , wherein the chimeric antigen receptor further comprises a spacer.

24. The method of claim 23 , wherein the spacer comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ TD NO: 16.

25. The method of claim 23 , wherein the spacer comprises a stalk region derived from at least one of a CD8alpha hinge domain, a CD28 hinge domain, a CTLA-4 hinge domain, an LNGFR extracellular domain, an IgG1 hinge, an IgG4 hinge, and a CH2-CH3 domain.

26. The method of claim 25 , wherein the stalk region comprises an amino acid sequence having at least about 90% identity with a CD8alpha hinge domain.

27. The method of claim 23 , wherein the spacer comprises: a stalk region; and a stalk extension region derived from at least one of a CD8alpha hinge domain, a CD28 hinge domain, a CTLA-4 hinge domain, an LNGFR extracellular domain, an IgG1 hinge, an IgG4 hinge, and a CH2-CH3 domain.

28. The method of claim 27 , wherein the stalk extension region comprises an amino acid sequence having at least about 90% identity with a CD8alpha hinge domain.

29. The method of claim 23 , wherein the spacer comprises a stalk region having one or more dimerization sites.

30. The method of claim 29 , wherein the spacer further comprises a stalk extension region lacking one or more dimerization sites as compared to the stalk region.

31. The method of claim 30 , wherein the stalk extension region lacks a dimerization site.

32. The method of claim 1 , wherein the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 30-33.

33. The method of claim 32 , wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 30 or 31.

34. The method of claim 32 , wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 31.

35. The method of claim 32 , wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 30.

36. The method of claim 1 , wherein the vector further encodes a cell tag.

37. The method of claim 36 , wherein the cell tag comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 66.

38. The method of claim 36 , wherein the cell tag is a HER1, a truncated HER1, a CD20, or a truncated CD20.

39. The method of claim 38 , wherein the truncated HER1 is capable of binding cetuximab but does not bind EGF ligand, does not allow for homo- and hetero-dimerization of EGFR, and does not allow for EGFR-mediated signaling.

40. The method of claim 1 , wherein the genetically-engineered cell is cryopreserved or infused within 0.5 hours into the subject following transfection.

41. The method of claim 1 , wherein the transmembrane domain is a synthetic linker or derived from the transmembrane region(s) of: the alpha, beta, or zeta chain of the T-cell receptor; CD28: CD3 epsilon; CD45; CD4; CD5: CD8alpha; CD9; CD16; CD22; CD33; CD37; CD64; CD80: CD86; CD134: CD137; or CD154.

42. The method of claim 41 , wherein the transmembrane domain is derived from a CD8alpha or CD28 transmembrane domain.

43. The method of claim 42 , wherein the transmembrane domain comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 21.

44. The method of claim 1 , wherein the cytoplasmic domain comprises a cytoplasmic signaling sequence derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, and/or CD66d.

45. The method of claim 1 , wherein the vector further encodes a self-cleaving Thosea asigna virus (12A) peptide.

46. The method of claim 1 , wherein the vector comprises a promoter.

47. The method of claim 46 , wherein the promoter is a hEF1a1 promoter.

48. The method of claim 1 , wherein the cell is a T cell, a Natural Killer (NK) cell, or a cytotoxic T lymphocyte (CTL).

49. The method of claim 48 , wherein the cell is a T cell or a NK cell.

50. The method of claim 49 , wherein the cell is a T cell.

51. The method of claim 1 , wherein, following transfection, the cell is cultured ex vivo for 48 hours or less.

52. The method of claim 1 , wherein the vector comprises a recombination site.

53. The method of claim 52 , wherein the recombination site is an AttP or AttB recombination sequence.

54. The method of claim 53 , wherein the recombination site is an AttP recombination site.

55. The method of claim 53 , wherein the recombination site is an AttB recombination site.

56. The method of claim 52 , wherein the step of transfecting the cell involves introducing into the cell a site-specific recombinase or a polynucleotide encoding such a recombinase.

57. The method of claim 52 , wherein the recombinase is from the integrase or resolvase family.

58. The method of claim 57 , wherein the recombinase is a serine recombinase.

59. The method of claim 58 , wherein the serine recombinase is a Listeria monoytogenes phage recombinase, a Streptococcus pyogenes phage recombinase, a Bacillus subtilis phage recombinase, a Mycobacterium tuberculosis phage recombinase, or a Mycobacterium smegmatis phage recombinase.

60. The method of claim 58 , wherein the serine recombinase is a SF370, SPβc2, Bxb1, A118, or φRv1 serine recombinase.

61. The method of claim 60 , wherein the serine recombinase is an SF370 recombinase.

62. The method of claim 60 , wherein the serine recombinase is an SPβc2 recombinase.

63. The method of claim 1 , wherein the chimeric antigen receptor further comprises:

(a) a spacer region comprising a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 16;

(b) a transmembrane domain comprising a polypeptide comprising a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 20; and

(c) a cytoplasmic comprising: (i) a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 22; and (ii) a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 26.

64. The method of claim 63 , wherein the vector further encodes:

(a) a cell tag comprising a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 66; and

(b) a fusion protein comprising a polypeptide having at least 95% identity with the amino acid sequence of SEQ ID NO: 69.

65. The method of claim 1 , wherein the chimeric antigen receptor comprises:

(a) a spacer region comprising a polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 16;

(b) a transmembrane domain comprising polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 20; and

(c) a cytoplasmic domain comprising: (i) a polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 22; and (ii) a polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 26.

66. The method of claim 65 , wherein the vector further encodes:

(a) a cell tag comprising a polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 66; and

(b) a fusion protein comprising a polypeptide having at least 98% identity with the amino acid sequence of SEQ ID NO: 69.

67. The method of claim 1 , wherein the chimeric antigen receptor further comprises:

(a) a spacer region comprising a polypeptide having at least 99% identity with the amino acid sequence of SEQ ID NO: 16;

(b) a transmembrane domain comprising a polypeptide having at least 99% identity with the amino acid sequence of SEQ ID NO: 20; and

(c) a cytoplasmic domain comprising: (i) a polypeptide having at least 99% identity with the amino acid sequence of SEQ ID NO: 22; and (ii) a polypeptide having at least 99% identity with the amino acid sequence of SEQ ID NO: 26.

68. The method of claim 67 , wherein the vector further encodes:

(a) a cell tag comprising a polypeptide having at least 99% identity with the amino acid sequence of SEQ ID NO: 66; and

(b) a fusion protein comprising a polypeptide having at least 99% identity with the amino acid sequence of SEQ ID NO: 69.

69. The method of claim 1 , wherein the chimeric antigen receptor comprises:

(a) a spacer region comprising the amino acid sequence of SEQ ID NO: 16;

(b) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 20; and

(c) a cytoplasmic domain comprising: (i) the amino acid sequence of SEQ ID NO: 22; and (ii) the amino acid sequence of SEQ ID NO: 26.

70. The method of claim 69 , wherein the vector further encodes:

(a) a cell tag comprising the amino acid sequence of SEQ ID NO: 66; and

(b) a fusion protein comprising the amino acid sequence of SEQ ID NO: 69.