MUC16 specific chimeric antigen receptors and uses thereof
Provided herein are chimeric antigen receptors (CARs) for cancer therapy, and more particularly, CARs containing a scFv from an anti-MUC16 monoclonal antibody. Provided are immune effector cells containing such CARs, and methods of treating proliferative disorders.
1. A nucleic acid encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises:
(a) an antigen-binding domain comprising: (i) the amino acid sequence of SEQ ID NO: 5; and (ii) the amino acid sequence of SEQ ID NO: 6;
(b) a transmembrane domain; and
(c) a cytoplasmic domain.
2. The nucleic acid of claim 1 , wherein the chimeric antigen receptor comprises the amino acid sequence of any one of SEQ ID NOs: 30-33.
3. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises a 4-1BB costimulatory signaling domain comprising a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 22.
4. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 26.
5. The nucleic acid of claim 1 , further encoding a cell tag.
6. The nucleic acid of claim 5 , wherein the nucleic acid encodes a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 66.
7. The nucleic acid of claim 5 , wherein the cell tag is a HER1, a truncated HER1, a CD20, or a truncated CD20.
8. The nucleic acid of claim 7 , 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.
9. A vector comprising a backbone and the nucleic acid of claim 1 .
10. The vector of claim 9 , wherein the vector is a lentiviral vector, a retroviral vector, or a non-viral vector.
11. The vector of claim 10 , wherein the vector is a non-viral vector.
12. A system for expressing a chimeric antigen receptor in an immune effector cell, the system comprising the vector of claim 9 .
13. The system of claim 12 , wherein the vector is a Sleeping Beauty Tc1/mariner-type transposon.
14. The system of claim 13 , further comprising a Sleeping Beauty Tc1/mariner-type transposase.
15. The system of claim 14 , wherein the Sleeping Beauty Tc1/mariner-type transposase is SB11, SB100X or SB110.
16. The system of claim 15 , wherein the Sleeping Beauty Tc 1/mariner-type transposase is SB11.
17. A method of expressing a chimeric antigen receptor in an immune effector cell, the method comprising contacting the immune effector cell with the vector of claim 9 .
18. The method of claim 17 , further comprising introducing into the immune effector cell a site-specific recombinase or a polynucleotide encoding such a recombinase.
19. The method of claim 18 , wherein the recombinase is from the integrase or resolvase family.
20. The method of claim 19 , wherein the recombinase is a serine recombinase.
21. The method of claim 20 , wherein the serine recombinase is a Listeria monocytogenes phage recombinase, a Streptococcus pyogenes phage recombinase, a Bacillus subtilis phage recombinase, a Mycobacterium tuberculosis phage recombinase, or a Mycobacterium smegmatis phage recombinase.
22. The method of claim 20 , wherein the serine recombinase is a SF370, SPβc2, Bxb1, A118, or φRvl serine recombinase.
23. The method of claim 22 , wherein the serine recombinase is an SF370 recombinase.
24. The method of claim 22 , wherein the serine recombinase is an SPβc2 recombinase.
25. The vector of claim 9 , further encoding a cytokine.
26. The vector of claim 25 , 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, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, or a functional fragment or variant of any of the foregoing.
27. The vector of claim 26 , wherein the cytokine is IL-15, or a functional fragment or variant thereof.
28. The vector of claim 9 , further encoding a fusion protein comprising: IL-15, or a functional fragment or variant thereof; and IL-15Ra, or a functional fragment or variant thereof.
29. The vector of claim 28 , wherein the fusion protein comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 69.
30. The vector of claim 9 , further encoding a self-cleaving Thosea asigna virus (T2A) peptide.
31. The vector of claim 9 , further comprising a promoter.
32. The vector of claim 31 , wherein the promoter is a hEF1a1 promoter.
33. The vector of claim 9 , comprising a recombination site.
34. The vector of claim 33 , wherein the recombination site is an AttP or AttB recombination sequence.
35. The vector of claim 34 , wherein the recombination site is an AttP recombination site.
36. The vector of claim 34 , wherein the recombination site is an AttB recombination site.
37. The vector of claim 9 , wherein the chimeric antigen receptor 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 having at least 95% identity with the amino acid sequence of SEQ ID NO: 20; and
(c) a cytoplasmic domain 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.
38. The vector of claim 37 , further encoding:
(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.
39. The vector of claim 9 , 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 a 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.
40. The vector of claim 39 , 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.
41. The vector of claim 9 , 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) 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.
42. The vector of claim 41 , 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.
43. The vector of claim 9 , 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.
44. The vector of claim 43 , further encoding:
(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.
45. A system for expressing a chimeric antigen receptor in an immune effector cell, the system comprising the vector of claim 43 .
46. A method of expressing a chimeric antigen receptor in an immune effector cell, the method comprising contacting the immune effector cell with the vector of claim 43 .
47. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a human subject in need thereof, the method comprising administering to the human subject an effective amount of an immune effector cell produced by introducing the vector of claim 9 into a cell.
48. The method of claim 47 , wherein the immune effector cell is cryopreserved or immediately infused within 0.5 hours into the subject following transfection.
49. The method of claim 47 , wherein, following introduction of the vector, the immune effector cell is propagated ex vivo for 48 hours or less.
50. The nucleic acid 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.
51. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises a cytoplasmic signaling sequence derived from CD3 zeta.
52. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises a 4-1BB and/or a CD28 costimulatory signaling domain.
53. The nucleic acid of claim 1 , wherein the chimeric antigen receptor further comprises a spacer.
54. The nucleic acid of claim 53 , wherein the spacer comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 16.
55. The nucleic acid of claim 53 , 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.
56. The nucleic acid of claim 55 , wherein the stalk region comprises an amino acid sequence having at least about 90% identity with a CD8alpha hinge domain.
57. The nucleic acid of claim 53 , wherein the spacer comprises: (a) a stalk region; and (b) 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.
58. The nucleic acid of claim 57 , wherein the stalk extension region comprises an amino acid sequence having at least about 90% identity with a CD8alpha hinge domain.
59. The nucleic acid of claim 53 , wherein the spacer comprises a stalk region having one or more dimerization sites.
60. The nucleic acid of claim 59 , wherein the spacer further comprises a stalk extension region lacking one or more dimerization sites as compared to the stalk region.
61. The nucleic acid of claim 60 , wherein the stalk extension region lacks a dimerization site.
62. The nucleic acid of claim 1 , wherein the transmembrane domain is a synthetic linker or is derived from 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.
63. The nucleic acid of claim 62 , wherein the transmembrane domain is derived from a CD8alpha or CD28 transmembrane domain.
64. The nucleic acid of claim 63 , wherein the transmembrane domain comprises a polypeptide having at least 90% identity with the amino acid sequence of SEQ ID NO: 21.
65. The nucleic acid 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.
66. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises: (a) a CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, DAP10, and/or DAP12 costimulatory signaling domain; and/or (b) a ligand that specifically binds with CD83.
67. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises a 4-1BB and a CD28 costimulatory signaling domain.
68. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises a 4-1BB costimulatory signaling domain.
69. The nucleic acid of claim 1 , wherein the cytoplasmic domain comprises a CD28 costimulatory signaling domain.
70. The nucleic acid of claim 1 , wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 30 or 31.
71. The nucleic acid of claim 70 , wherein the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 30.
72. The nucleic acid 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.
73. An immune effector cell comprising a chimeric antigen receptor comprising:
(a) an antigen-binding domain comprising: (i) the amino acid sequence of SEQ ID NO: 5; and (ii) the amino acid sequence of SEQ ID NO: 6;
(b) a transmembrane domain; and
(c) a cytoplasmic domain.
74. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a human subject in need thereof the method comprising administering to the human subject an effective amount of the cell of claim 73 .
75. The immune effector cell of claim 73 , further comprising a fusion protein comprising; IL-15, or a functional fragment or variant thereof; and IL-15Rα, or a functional fragment or variant thereof.
76. The immune effector cell of claim 73 , wherein the cell is a T cell, a Natural Killer (NK) cell, or a cytotoxic T lymphocyte (CTL).
77. The immune effector cell of claim 76 , wherein the cell is a T cell or a NK cell.
78. The immune effector cell of claim 77 , wherein the cell is a T cell.
79. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the cell of claim 73 .
80. The immune effector cell of claim 73 , 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.
81. A method for stimulating a T cell-mediated immune response to a target cell population or tissue in a human subject in need thereof, the method comprising administering to the human subject an effective amount of the cell of claim 80 .