Chimeric antigen receptors targeting G-protein coupled receptor and uses thereof
The presently disclosed subject matter provides for methods and compositions for treating multiple myeloma. It relates to chimeric antigen receptors (CARs) that specifically target a G-protein coupled receptor (e.g., a G-protein coupled receptor family C group 5 member D (GPRC5D)), and immunoresponsive cells comprising such CARs. The presently disclosed CARs targeting a G-protein coupled receptor (e.g., GPRC5D) have enhanced immune-activating properties, including anti-tumor activity.
1. A chimeric antigen receptor (CAR), comprising an extracellular antigen-binding domain that binds to a G-protein coupled receptor family C group 5 member D (GPRC5D), a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 220, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:221 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 222; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:224 and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:225.
2. The CAR of claim 1 , wherein the extracellular antigen-binding domain of the CAR binds to the GPRC5D with a binding affinity (K d ) of from about 1×10 −9 M to about 3×10 −6 M.
3. The CAR of claim 1 , wherein the GPRC5D is a human GPRC5D.
4. The CAR of claim 3 , wherein the extracellular antigen-binding domain binds to the human GPRC5D with a binding affinity (K d ) of from about 1×10 −9 M to about 1×10 −8 M.
5. The CAR of claim 1 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
6. The CAR of claim 5 , wherein the extracellular antigen-binding domain is a human scFv.
7. The CAR of claim 1 , wherein the extracellular antigen-binding domain is a Fab.
8. The CAR of claim 1 , wherein the heavy chain variable region sequence comprises the amino acid sequence set forth in SEQ ID NO: 65.
9. The CAR of claim 1 , wherein the light chain variable region sequence comprises the amino acid sequence set forth in SEQ ID NO: 66.
10. The CAR of claim 1 , wherein:
the heavy chain variable region comprises an amino acid sequence that is at least about 90% homologous to the amino acid sequence set forth in SEQ ID NO:65, and the light chain variable region comprises an amino acid sequence that is at least about 90% homologous to the amino acid sequence set forth in SEQ ID NO:66.
11. The CAR of claim 10 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
12. The CAR of claim 1 , wherein:
the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:65, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:66.
13. The CAR of claim 12 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
14. The CAR of claim 1 , wherein the extracellular antigen-binding domain comprises a human variable region framework region.
15. The CAR of claim 5 , wherein the scFv comprises the amino acid sequence set forth in SEQ ID NO: 116.
16. The CAR of claim 1 , wherein the extracellular antigen-binding domain binds to at least one of:
an epitope region comprising amino acids 15-23 of SEQ ID NO:97 and
an epitope region comprising amino acids 230-243 of SEQ ID NO:97.
17. The CAR of claim 1 , wherein the transmembrane domain comprises a transmembrane domain of 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 that is not based on a protein associated with the immune response, or a combination thereof.
18. The CAR of claim 17 , wherein the transmembrane domain comprises a transmembrane domain of a CD8 polypeptide or a CD28 polypeptide.
19. The CAR of claim 1 , wherein the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide.
20. The CAR of claim 19 , wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling region.
21. The CAR of claim 12 , wherein the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide.
22. The CAR of claim 21 , wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling region.
23. The CAR of claim 20 , wherein the at least one co-stimulatory signaling region comprises an intracellular signaling region of a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof.
24. The CAR of claim 23 , wherein the at least one co-stimulatory signaling region comprises an intracellular signaling region of a CD28 polypeptide or a 4-1BB polypeptide.
25. The CAR of claim 22 , wherein the at least one co-stimulatory signaling region comprises an intracellular signaling region of a CD28 polypeptide or a 4-1BB polypeptide.
26. The CAR of claim 20 , wherein
(a) the transmembrane domain comprises a transmembrane domain of a CD28 polypeptide, and the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide and at least one co-stimulatory signaling region that comprises an intracellular signaling region of a CD28 polypeptide; or
(b) the transmembrane domain comprises a transmembrane domain of a CD8 polypeptide, and the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide and at least one co-stimulatory signaling region that comprises an intracellular signaling region of a 4-1BB polypeptide.
27. The CAR of claim 1 , wherein
(a) the transmembrane domain comprises a transmembrane domain of a CD28 polypeptide, and the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide and at least one co-stimulatory signaling region that comprises an intracellular signaling region of a 4-1BB polypeptide.
28. The CAR of claim 27 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
29. The CAR of claim 12 , wherein
(a) the transmembrane domain comprises a transmembrane domain of a CD28 polypeptide, and the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide and at least one co-stimulatory signaling region that comprises an intracellular signaling region of a CD28 polypeptide; or
(b) the transmembrane domain comprises a transmembrane domain of a CD8 polypeptide, and the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide and at least one co-stimulatory signaling region that comprises an intracellular signaling region of a 4-1BB polypeptide.
30. The CAR of claim 29 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
31. An immunoresponsive cell comprising the CAR of claim 1 .
32. The immunoresponsive cell of claim 31 , wherein the immunoresponsive cell is further transduced with (a) at least one co-stimulatory ligand such that the immunoresponsive cell expresses the at least one co-stimulatory ligand; and/or (b) at least one cytokine such that the immunoresponsive cell secretes the at least one cytokine.
33. The immunoresponsive cell of claim 31 , 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.
34. A T cell, wherein the T cell comprises the CAR of claim 1 .
35. An immunoresponsive cell, wherein the immunoresponsive cell comprises the CAR of claim 27 .
36. A T cell, wherein the T cell comprises the CAR of claim 27 .
37. An immunoresponsive cell, wherein the immunoresponsive cell comprises the CAR of claim 29 .
38. A T cell, wherein the T cell comprises the CAR of claim 29 .
39. A composition comprising an effective amount of the immunoresponsive cells of claim 31 .
40. A composition comprising an effective amount of the T cells of claim 34 .
41. The composition of claim 39 , which is a pharmaceutical composition that further comprises a pharmaceutically acceptable excipient.
42. A kit for treating a neoplasia, comprising the immunoresponsive cell of claim 31 and written instructions for using the immunoresponsive cells for treating a subject having a neoplasia selected from multiple myeloma and Waldenstrom's Macroglobulinemia.
43. The CAR of claim 26 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
44. The CAR of claim 12 , wherein the transmembrane domain comprises a transmembrane domain of a CD28 polypeptide, and the intracellular signaling domain comprises a signaling domain of a CD3 polypeptide and at least one co-stimulatory signaling region that comprises an intracellular signaling region of a 4-1BB polypeptide.
45. The CAR of claim 44 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
46. An immunoresponsive cell, wherein the immunoresponsive cell comprises the CAR of claim 44 .
47. A T cell, wherein the T cell comprises the CAR of claim 44 .
48. A composition comprising an effective amount of the immunoresponsive cells of claim 46 .
49. A composition comprising an effective amount of the T cells of claim 47 .