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 (GPRCSD), a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises: (i) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:202, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:204; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:205, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:206, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:207; (ii) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:208, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:209, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:210; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:211, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:212, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:213; (iii) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:214, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:215, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:216; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:217, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:218, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:219; or (iv) a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:226, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:227, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:228; and a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:229, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:230, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:231.
2. The CAR of claim 1 , wherein the extracellular antigen-binding domain 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 GPRC5D with a binding affinity (K d ) of from about 1×10 −9 M to about 3×10 −6 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 scFv comprises the amino acid sequence set forth in SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, or SEQ ID NO:117.
7. The CAR of claim 5 , wherein the extracellular antigen-binding domain is a human scFv.
8. The CAR of claim 1 , wherein the extracellular antigen-binding domain is an antigen-binding fragment (Fab).
9. The CAR of claim 1 , wherein the extracellular antigen-binding domain comprises a human variable region framework region.
10. The CAR of claim 1 , wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, or SEQ ID NO:69.
11. The CAR of claim 1 , wherein the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, or SEQ ID NO:70.
12. The CAR of claim 1 , wherein: the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:53, SEQ ID NO:57, SEQ ID NO:61, or SEQ ID NO:69; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:54, SEQ ID NO:58, SEQ ID NO:62, or SEQ ID NO:70.
13. The CAR of claim 1 , wherein: (i) 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:53, and 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:54; (ii) 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:57, and 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:58; (iii) 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:61, and 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:62; or (iv) 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:69, and 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:70.
14. The CAR of claim 13 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
15. The CAR of claim 1 , wherein: (i) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:53, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:54; (ii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:57, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:58; (iii) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:61, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:62; or (iv) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:69, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:70.
16. The CAR of claim 15 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
17. The CAR of claim 15 , wherein the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide.
18. The CAR of claim 17 , wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling region.
19. The CAR of claim 18 , wherein the at least one co-stimulatory signaling region comprises an intracellular signaling region of a CD28 polypeptide or a 4-1BB polypeptide.
20. The CAR of claim 15 , 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.
21. The CAR of claim 20 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
22. The CAR of claim 15 , wherein 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.
23. The CAR of claim 22 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
24. The CAR of claim 1 , wherein the extracellular antigen-binding domain binds to an epitope region selected from the group consisting of:
an epitope region comprising amino acids 16-23 of SEQ ID NO:97,
an epitope region comprising amino acids 15-23 of SEQ ID NO:97,
an epitope region comprising amino acids 16-25 of SEQ ID NO:97,
an epitope region comprising amino acids 10-17 of SEQ ID NO:97,
an epitope region comprising amino acids 5-17 of SEQ ID NO:97,
an epitope region comprising amino acids 85-95 of SEQ ID NO:97,
an epitope region comprising amino acids 157-164 of SEQ ID NO:97,
an epitope region comprising amino acids 157-167 of SEQ ID NO:97,
an epitope region comprising amino acids 230-237 of SEQ ID NO:97,
an epitope region comprising amino acids 229-237 of SEQ ID NO:97,
an epitope region comprising amino acids 230-243 of SEQ ID NO:97, and
an epitope region comprising amino acids 227-237 of SEQ ID NO:97.
25. 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, or a combination thereof.
26. The CAR of claim 25 , wherein the transmembrane domain comprises a transmembrane domain of a CD8 polypeptide or a CD28 polypeptide.
27. The CAR of claim 1 , wherein the intracellular signaling domain comprises a signaling domain of a CD3ζ polypeptide.
28. The CAR of claim 27 , wherein the intracellular signaling domain further comprises at least one co-stimulatory signaling region.
29. The CAR of claim 28 , 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.
30. The CAR of claim 29 , wherein the at least one co-stimulatory signaling region comprises an intracellular signaling region of a CD28 polypeptide or a 4-1BB polypeptide.
31. The CAR of claim 28 , 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.
32. The CAR of claim 31 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
33. The CAR of claim 1 , wherein 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.
34. The CAR of claim 33 , wherein the extracellular antigen-binding domain is a single-chain variable fragment (scFv).
35. An immunoresponsive cell comprising the CAR of claim 1 .
36. The immunoresponsive cell of claim 35 , 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.
37. The immunoresponsive cell of claim 35 , 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.
38. An immunoresponsive cell, wherein the immunoresponsive cell comprises the CAR of claim 20 .
39. An immunoresponsive cell, wherein the immunoresponsive cell comprises the CAR of claim 22 .
40. An immunoresponsive cell, wherein the immunoresponsive cell comprises the CAR of claim 33 .
41. A T cell, wherein the T cell comprises the CAR of claim 1 .
42. A T cell, wherein the T cell comprises the CAR of claim 20 .
43. A T cell, wherein the T cell comprises the CAR of claim 22 .
44. A T cell, wherein the T cell comprises the CAR of claim 33 .
45. A composition comprising the immunoresponsive cell of claim 35 .
46. The composition of claim 45 , further comprising a pharmaceutically acceptable excipient.
47. A composition comprising the immunoresponsive cell of claim 39 .
48. A composition comprising the T cell of claim 41 .
49. A composition comprising the T cell of claim 43 .
50. A kit for treating a neoplasia, comprising the immunoresponsive cell of claim 35 and written instructions for using the immunoresponsive cell for treating a subject having a neoplasia selected from multiple myeloma and Waldenstrom's Macroglobulinemia.