Chimeric antigen receptors targeting Fc receptor-like 5 and uses thereof
The presently disclosed subject matter provides for methods and compositions for treating a neoplasia (e.g., multiple myeloma). It relates to chimeric antigen receptors (CARs) that specifically target Fc Receptor-like 5 (FcRL5), e.g., domain 9 of FcRL5, and immunoresponsive cells comprising such CARs. The presently disclosed FcRL5-targeted CARs have enhanced immune-activating properties, including anti-tumor activity.
1. A chimeric antigen receptor (CAR), comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain specifically binds to Fc Receptor-Like 5 (FcRL5) and comprises:
(a) a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region sequence set forth in SEQ ID NO:143, and a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region sequence set forth in SEQ ID NO:144;
(b) a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region sequence set forth in SEQ ID NO:215, and a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region sequence set forth in SEQ ID NO:216;
(c) a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region sequence set forth in SEQ ID NO:267, and a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region sequence set forth in SEQ ID NO:268; or
(d) a light chain variable region comprising a CDR1, a CDR2, and a CDR3 of the light chain variable region sequence set forth in SEQ ID NO:171, and a heavy chain variable region comprising a CDR1, a CDR2, and a CDR3 of the heavy chain variable region sequence set forth in SEQ ID NO:172.
2. The CAR of claim 1 , wherein the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv).
3. The CAR of claim 2 , wherein the extracellular antigen-binding domain comprises a human scFv.
4. The CAR of claim 2 , wherein the scFv is comprised in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain.
5. The CAR of claim 1 , wherein the extracellular antigen-binding domain of the CAR binds to FcRL5 with a binding affinity (K d ) of from about 1×10 −9 M to about 3×10 −6 M.
6. The CAR of claim 1 , wherein the extracellular antigen-binding domain of the CAR binds to domain 7, domain 8 or domain 9 of FcRL5 with a binding affinity (K d ) of from about 1×10 −9 M to about 3×10 −6 M.
7. The CAR of claim 1 wherein the light chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 318, a CDR2 comprising the sequence set forth in SEQ ID NO: 319, and a CDR3 comprising the sequence set forth in SEQ ID NO: 419, and the heavy chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 411, a CDR2 comprising the sequence set forth in SEQ ID NO: 412, and a CDR3 comprising the sequence set forth in SEQ ID NO: 463.
8. The CAR of claim 1 , wherein:
(a) the light chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:143, and the heavy chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:144;
(b) the light chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:215, and the heavy chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:216;
(c) the light chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:267, and the heavy chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:268; or
(d) the light chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:171, and the heavy chain variable region comprises an amino acid sequence that has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:172.
9. The CAR of claim 8 , wherein:
(a) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:143, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:144;
(b) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:215, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:216;
(c) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:267, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:268; or
(d) the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:171, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:172.
10. The CAR of claim 9 , wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:143, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:144.
11. The CAR of claim 9 , wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:215, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:216.
12. The CAR of claim 9 , wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:267, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:268.
13. The CAR of claim 9 , wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:171, and the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:172.
14. The CAR of claim 9 , wherein the extracellular antigen-binding domain comprises the amino acid sequence set forth in SEQ ID NO:664, SEQ ID NO:700, SEQ ID NO:726, or SEQ ID NO:678.
15. The CAR of claim 7 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
16. The CAR of claim 1 , wherein the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide, or a combination thereof.
17. The CAR of claim 16 , wherein the transmembrane domain comprises a CD8 polypeptide or a CD28 polypeptide.
18. The CAR of claim 1 , wherein the intracellular domain comprises a CD3ζ polypeptide.
19. The CAR of claim 18 , wherein the intracellular domain further comprises at least one signaling region.
20. The CAR of claim 19 , wherein the at least one signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide, or a combination thereof.
21. The CAR of claim 19 , wherein the at least one signaling region comprises at least one co-stimulatory signaling region.
22. The CAR of claim 21 , wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.
23. The CAR of claim 21 , wherein the at least one co-stimulatory signaling region comprises a CD28 polypeptide or a 4-1BB polypeptide.
24. The CAR of claim 23 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the at least one co-stimulatory signaling region comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the at least one co-stimulatory signaling region comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, the intracellular domain comprises a CD3ζ polypeptide, and the at least one co-stimulatory signaling region comprises a 4-1BB polypeptide.
25. The CAR of claim 10 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
26. An immunoresponsive cell comprising the CAR of claim 1 .
27. The immunoresponsive cell of claim 26 , wherein the CAR is constitutively expressed on the surface of the immunoresponsive cell.
28. The immunoresponsive cell of claim 26 , wherein the immunoresponsive cell is further transduced with a nucleic acid molecule encoding at least one co-stimulatory ligand such that the immunoresponsive cell expresses the at least one co-stimulatory ligand; or a nucleic acid molecule encoding at least one cytokine such that the immunoresponsive cell secretes the at least one cytokine.
29. The immunoresponsive cell of claim 26 , wherein the immunoresponsive cell is selected from the group consisting of a T cell, a Natural Killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a human embryonic stem cell, a lymphoid progenitor cell, a T cell-precursor cell, and a pluripotent stem cell from which lymphoid cells may be differentiated.
30. A nucleic acid molecule encoding the CAR of claim 1 .
31. The nucleic acid molecule of claim 30 , comprising the nucleotide sequence set forth in SEQ ID NO: 954, SEQ ID NO: 956, SEQ ID NO: 959, or SEQ ID NO: 955, or a sequence having at least about 90% identity to the nucleotide sequence set forth in SEQ ID NO:954, SEQ ID NO:956, SEQ ID NO:959, or SEQ ID NO:955.
32. A vector comprising the nucleic acid molecule of claim 30 .
33. A host cell expressing the nucleic acid molecule of claim 30 .
34. A T cell comprising the CAR of claim 1 .
35. A method for producing an immunoresponsive cell that binds to Fc Receptor-Like 5 (FcRL5), comprising introducing into an immunoresponsive cell a nucleic acid molecule that encodes the CAR of claim 1 .
36. A pharmaceutical composition comprising an effective amount of the immunoresponsive cell of claim 10 and a pharmaceutically acceptable excipient.
37. A kit for treating a tumor, the kit comprising the immunoresponsive cell of claim 10 and written instructions for using the immunoresponsive cell for treating a subject having a tumor.
38. The kit of claim 37 , wherein the tumor is selected from the group consisting of multiple myeloma, Non-Hodgkin Lymphoma, Mantle Cell Lymphoma, Hodgkin Lymphoma, Chronic Lymphocytic Leukemia (CLL), Acute lymphocytic leukemia (ALL), Hairy Cell Leukemia, Burkitt's Lymphoma, and Waldenstrom's Macroglobulinemia.
39. The CAR of claim 11 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
40. The CAR of claim 12 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
41. The CAR of claim 13 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
42. A nucleic acid molecule encoding the CAR of claim 7 .
43. A vector comprising the nucleic acid molecule of claim 42 .
44. An immunoresponsive cell comprising the CAR of claim 7 .
45. A T cell comprising the CAR of claim 7 .
46. The CAR of claim 1 , wherein the light chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 318, a CDR2 comprising the sequence set forth in SEQ ID NO: 319, and a CDR3 comprising the sequence set forth in SEQ ID NO: 531, and the heavy chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 515, a CDR2 comprising the sequence set forth in SEQ ID NO: 516, and a CDR3 comprising the sequence set forth in SEQ ID NO: 517.
47. The CAR of claim 1 , wherein the light chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 571, a CDR2 comprising the sequence set forth in SEQ ID NO: 572, and a CDR3 comprising the sequence set forth in SEQ ID NO: 573, and the heavy chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 372, a CDR2 comprising the sequence set forth in SEQ ID NO: 475, and a CDR3 comprising the sequence set forth in SEQ ID NO: 570.
48. The CAR of claim 1 , wherein the light chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 490, a CDR2 comprising the sequence set forth in SEQ ID NO: 313, and a CDR3 comprising the sequence set forth in SEQ ID NO: 491, and the heavy chain variable region comprises a CDR1 comprising the sequence set forth in SEQ ID NO: 309, a CDR2 comprising the sequence set forth in SEQ ID NO: 310, and a CDR3 comprising the sequence set forth in SEQ ID NO: 489.
49. The CAR of claim 46 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
50. The CAR of claim 47 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
51. The CAR of claim 48 , wherein:
(a) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a CD28 polypeptide;
(b) the transmembrane domain comprises a CD8 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide; or
(c) the transmembrane domain comprises a CD28 polypeptide, and the intracellular domain comprises a CD3ζ polypeptide and a co-stimulatory signaling region that comprises a 4-1BB polypeptide.
52. A nucleic acid molecule encoding the CAR of claim 46 .
53. A vector comprising the nucleic acid molecule of claim 52 .
54. An immunoresponsive cell comprising the CAR of claim 46 .
55. A T cell comprising the CAR of claim 46 .
56. A nucleic acid molecule encoding the CAR of claim 47 .
57. A vector comprising the nucleic acid molecule of claim 56 .
58. An immunoresponsive cell comprising the CAR of claim 47 .
59. A T cell comprising the CAR of claim 47 .
60. A nucleic acid molecule encoding the CAR of claim 48 .
61. A vector comprising the nucleic acid molecule of claim 60 .
62. An immunoresponsive cell comprising the CAR of claim 48 .
63. A T cell comprising the CAR of claim 48 .