Chimeric antigen receptor specific for GPRC5D and BCMA

Abstract

Chimeric antigen receptors (CARs) are provided that contain an extracellular antigen-binding domain that binds to G protein-coupled receptor class C group 5 member D (GPRC5D) and B cell maturation antigen (BCMA). The present disclosure further relates to genetically engineered cells that express such CARs and their use in adoptive cell therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 63 / 395,702, entitled "CHIMERIC ANTIGEN RECEPTORS SPECIFIC FOR GPRC5D AND BCMA," filed August 5, 2022, the contents of which are incorporated by reference in their entirety.

[0002] Field In some embodiments, the present disclosure relates to chimeric antigen receptors (CARs), which contain an extracellular antigen-binding domain that binds to G protein-coupled receptor class C group 5 member D (GPRC5D) and B-cell maturation antigen (BCMA). The present disclosure further relates to genetically engineered cells that express such CARs and their use in adoptive cell therapy.

[0003] Incorporation by reference of sequence listing This application is filed with a Sequence Listing in electronic format, which is provided as a file entitled "735042026340SeqList.xml," created on August 4, 2023, and 224,174 bytes in size. The information in the electronic Sequence Listing is incorporated by reference in its entirety. [Background technology]

[0004] background G protein-coupled receptor class C group 5 member D (GPRC5D) is a G protein-coupled receptor that is highly expressed in bone marrow samples from patients with multiple myeloma (MM), compared with low GPRC5D expression in bone marrow samples from patients with other hematological malignancies. B cell maturation antigen (BCMA) is a type III transmembrane protein expressed on mature B lymphocytes. Various GPRC5D-binding chimeric antigen receptors (CARs), BCMA-binding CARs, and cells expressing such CARs are available. However, there remains a need for improved CARs that bind to both GPRC5D and BCMA, and engineered cells expressing them for use in adoptive cell therapy, etc. Provided herein are embodiments that address this need. Summary of the Invention

[0005] Provided herein is a bispecific chimeric antigen receptor (CAR) comprising an extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA.

[0006] Also disclosed herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in amino to carboxy terminus: (i) one of the VH region and VL region of the GPRC5D-binding domain, one of the VH region and VL region of the BCMA-binding domain; (ii) comprising one of the VH and VL regions of the BCMA-binding domain, one of the VH and VL regions of the GPRC5D-binding domain, the other of the VH and VL regions of the GPRC5D-binding domain, and the other of the VH and VL regions of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0007] In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, (i). In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, a VH region of the GPRC5D-binding domain, a VH region of the BCMA-binding domain, a VL region of the BCMA-binding domain, and a VL region of the GPRC5D-binding domain.

[0008] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the GPRC5D-binding domain, the VH region of the BCMA-binding domain, the VL region of the BCMA-binding domain, and the VL region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0009] In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, a VH region of the GPRC5D-binding domain, a VL region of the BCMA-binding domain, a VH region of the BCMA-binding domain, and a VL region of the GPRC5D-binding domain.

[0010] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the GPRC5D-binding domain, the VL region of the BCMA-binding domain, the VH region of the BCMA-binding domain, and the VL region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0011] In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, a VL region of the GPRC5D-binding domain, a VH region of the BCMA-binding domain, a VL region of the BCMA-binding domain, and a VH region of the GPRC5D-binding domain.

[0012] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the GPRC5D-binding domain, the VH region of the BCMA-binding domain, the VL region of the BCMA-binding domain, and the VH region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0013] In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, a VL region of the GPRC5D-binding domain, a VL region of the BCMA-binding domain, a VH region of the BCMA-binding domain, and a VH region of the GPRC5D-binding domain.

[0014] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the GPRC5D-binding domain, the VL region of the BCMA-binding domain, the VH region of the BCMA-binding domain, and the VH region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0015] In some embodiments, the extracellular domain comprises, in order from amino to carboxy terminus, (ii). In some embodiments, the extracellular domain comprises, in order from amino to carboxy terminus, a VH region of the BCMA-binding domain, a VH region of the GPRC5D-binding domain, a VL region of the GPRC5D-binding domain, and a VL region of the BCMA-binding domain.

[0016] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the BCMA-binding domain, the VH region of the GPRC5D-binding domain, the VL region of the GPRC5D-binding domain, and the VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0017] In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, a VH region of the BCMA-binding domain, a VL region of the GPRC5D-binding domain, a VH region of the GPRC5D-binding domain, and a VL region of the BCMA-binding domain.

[0018] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the BCMA-binding domain, the VL region of the GPRC5D-binding domain, the VH region of the GPRC5D-binding domain, and the VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0019] In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, a VL region of the BCMA-binding domain, a VH region of the GPRC5D-binding domain, a VL region of the GPRC5D-binding domain, and a VH region of the BCMA-binding domain.

[0020] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the BCMA-binding domain, the VH region of the GPRC5D-binding domain, the VL region of the GPRC5D-binding domain, and the VH region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0021] In some embodiments, the extracellular domain comprises, from amino to carboxy terminus, a VL region of the BCMA-binding domain, a VL region of the GPRC5D-binding domain, a VH region of the GPRC5D-binding domain, and a VH region of the BCMA-binding domain.

[0022] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the BCMA-binding domain, the VL region of the GPRC5D-binding domain, the VH region of the GPRC5D-binding domain, and the VH region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0023] In some embodiments, (a) the VH and VL regions of the GPRC5D-binding domain; and (b) the VH and VL regions of the BCMA-binding domain are connected by a linker.

[0024] In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linker is 4 to 12 amino acids in length. In some embodiments, the linker is or comprises the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 22. In some embodiments, the linker is or comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the linker is or comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the linker is or comprises the amino acid sequence set forth in SEQ ID NO: 22.

[0025] In some embodiments, (a) the VH and VL regions of the GPRC5D-binding domain are linked by a linker; or (b) the VH and VL regions of the BCMA-binding domain are linked by a linker. In some embodiments, the VH and VL regions of the GPRC5D-binding domain are linked by a linker. In some embodiments, the VH and VL regions of the BCMA-binding domain are linked by a linker.

[0026] In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0027] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, (i) the VH region of the GPRC5D-binding domain, (ii) a linker set forth in SEQ ID NO: 21, (iii) the VL region of the BCMA-binding domain, (iv) a linker set forth in SEQ ID NO: 17, (v) the VH region of the BCMA-binding domain, (vi) a linker set forth in SEQ ID NO: 21, and (vii) the VL region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0028] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, one of the VH region and VL region of the BCMA-binding domain, the other of the VH region and VL region of the BCMA-binding domain, one of the VH region and VL region of the GPRC5D-binding domain, and the other of the VH region and VL region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0029] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the GPRC5D-binding domain, the VH region of the GPRC5D-binding domain, one of the VH region and VL region of the BCMA-binding domain, and the other of the VH region and VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0030] In some embodiments, the GPRC5D-binding region and the BCMA-binding region are linked by a linker. In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linker is 4 to 12 amino acids in length. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 24. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 19. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the VH region and VL region of the BCMA-binding domain are linked by a linker comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0031] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the GPRC5D-binding domain, the VL region of the GPRC5D-binding domain, one of the VH region and VL region of the BCMA-binding domain, and the other of the VH region and VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain, wherein the GPRC5D-binding domain and the BCMA-binding domain are linked by a linker comprising the sequence set forth in SEQ ID NO: 19 or 21.

[0032] In some embodiments, the VH region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively. In some embodiments, the VL region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In some embodiments, the VH region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively; and the VL region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively. In some embodiments, the VH region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:7. In some embodiments, the VL region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the VH region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and the VL region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the VH region of the GPRC5D-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the VL region of the GPRC5D-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the VH region of the GPRC5D-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 7; and the VL region of the GPRC5D-binding domain comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0033] In some embodiments, the VH region of the BCMA binding domain comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. In some embodiments, the VL region of the BCMA binding domain comprises CDR-1, CDR-2, and CDR-3, which comprise the amino acid sequences set forth in SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively. In some embodiments, the VH region of the BCMA binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the VL region of the BCMA binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the VH region of the BCMA binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; and the VL region of the BCMA binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the VH region of the BCMA binding domain comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the VL region of the BCMA binding domain comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the VH region of the BCMA binding domain comprises the amino acid sequence set forth in SEQ ID NO: 15; and the VL region of the BCMA binding domain comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0034] In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 77, 78, 79, and 80. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the extracellular domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 90.

[0035] In some embodiments, the spacer comprises at least a portion of an immunoglobulin or a variant thereof. In some embodiments, the spacer comprises a hinge region of an immunoglobulin or a variant thereof. In some embodiments, the immunoglobulin hinge region is an IgG4 hinge region. In some embodiments, the hinge region comprises a human IgG4 hinge region or a variant thereof.

[0036] In some embodiments, the spacer is less than 15 amino acids or less than about 15 amino acids in length. In some embodiments, the spacer is 12 to 15 amino acids in length. In some embodiments, the spacer is about 12 amino acids in length. In some embodiments, the spacer is about 13 amino acids in length. In some embodiments, the spacer is about 14 amino acids in length. In some embodiments, the spacer is about 15 amino acids in length. In some embodiments, the spacer comprises the amino acid sequence set forth in SEQ ID NO:25, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the spacer comprises the amino acid sequence set forth in SEQ ID NO:25. In some embodiments, the spacer comprises an immunoglobulin CH3 region. In some embodiments, the spacer is about 100 to 125 amino acids in length. In some embodiments, the spacer is about 119 amino acids in length. In some embodiments, the spacer comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the spacer comprises the amino acid sequence set forth in SEQ ID NO:26. In some embodiments, the spacer is 200 to 250 amino acids in length. In some embodiments, the spacer is 220 to 240 amino acids in length. In some embodiments, the spacer comprises an immunoglobulin hinge region, an immunoglobulin CH2 region or chimeric CH2 regions of different immunoglobulins, and an immunoglobulin CH3. In some embodiments, the spacer comprises an IgG4 hinge region or a variant thereof, a chimeric CH2 region comprising a portion of an IgG4 CH2 and a portion of an IgG2 CH2 (IgG2 / 4 CH2 region), and an IgG4 CH3 region. In some embodiments, the spacer comprises the amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:27. In some embodiments, the spacer comprises the amino acid sequence set forth in SEQ ID NO:27.

[0037] In some embodiments, the transmembrane domain is or comprises the transmembrane domain of CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or comprises the transmembrane domain of human CD4, human CD28, or human CD8. In some embodiments, the transmembrane domain is or comprises the transmembrane domain of human CD4. In some embodiments, the transmembrane domain is or comprises the transmembrane domain of human CD28. In some embodiments, the transmembrane domain is or comprises the transmembrane domain of human CD8. In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:28. In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO:28.

[0038] In some embodiments, the intracellular signaling domain is a domain derived from a T cell receptor (TCR) component or comprises an immunoreceptor tyrosine-based activation motif (ITAM). In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of the CD3 zeta chain. In some embodiments, the extracellular signaling domain comprises the cytoplasmic signaling domain of the human CD3 zeta chain. In some embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling region. In some embodiments, the costimulatory signaling region is located between the transmembrane region and the intracellular signaling domain. In some embodiments, the costimulatory signaling region comprises the intracellular signaling domain of a T cell costimulatory molecule, or a signaling portion thereof. In some embodiments, the costimulatory signaling region comprises the intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. In some embodiments, the costimulatory signaling region comprises the intracellular signaling domain of human CD28, human 4-1BB, or human ICOS. In some embodiments, the costimulatory signaling region comprises the intracellular signaling domain of 4-1BB or a signaling portion thereof. In some embodiments, the costimulatory signaling region comprises the intracellular signaling domain of human 4-1BB. In some embodiments, the costimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO:29, or an amino acid sequence having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, the costimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO:29.

[0039] In some embodiments, the CAR comprises an amino acid sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 98% sequence identity to any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44.

[0040] In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:31. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:32. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:33. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:34. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:38. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:39. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:40. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:41. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:42. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:43. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO:44.

[0041] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus: (i) the VH region of the GPRC5D-binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; (ii) a linker set forth in SEQ ID NO: 21; and (iii) CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. and CDR-3, (iv) a linker set forth in SEQ ID NO: 17; (v) a VH region of the BCMA binding domain, comprising CDR-1, CDR-2, and CDR-3 having the amino acid sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; (vi) a linker set forth in SEQ ID NO: 21; and (vii) a VL region of the GPRC5D binding domain, comprising CDR-1, CDR-2, and CDR-3 having the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; (b) a spacer comprising the amino acid sequence set forth in SEQ ID NO: 27; (c) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 28; and (d) an intracellular signaling domain comprising the amino acid sequences set forth in SEQ ID NOs: 29 and 30.

[0042] In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the bispecific CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 119.

[0043] Also provided herein is a bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, (i) a VL region of the BCMA-binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; (ii) a linker set forth in SEQ ID NO: 21; (iii) CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; (iv) a linker set forth in SEQ ID NO: 17; (v) a VH region of the GPRC5D-binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; (vi) a linker set forth in SEQ ID NO: 21; and (vii) a VH region of the BCMA-binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; (b) a spacer comprising the amino acid sequence set forth in SEQ ID NO: 27; (c) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 28; and (d) an intracellular signaling domain comprising the amino acid sequences set forth in SEQ ID NOs: 29 and 30.

[0044] In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the bispecific CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 120.

[0045] Also provided herein is a polynucleotide encoding any of the CARs provided herein. In some embodiments, the polynucleotide comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 105-120. Also provided herein is a polynucleotide comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 105-120. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 5. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 6. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 7. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 8. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 9. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 10. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 12. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 13. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 14. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 15. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 16. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 18. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 20. In some embodiments, the polynucleotide is optimized by splice site removal. In some embodiments, the polynucleotide is codon optimized for expression in human cells.In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 119. In some embodiments, the polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO:120.

[0046] Also provided herein is a vector comprising any of the polynucleotides provided herein. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector or an adeno-associated viral (AAV) vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated viral (AAV) vector.

[0047] Also provided herein are cells comprising any of the CARs provided herein.

[0048] Also provided herein are cells comprising any of the polynucleotides provided herein.

[0049] Also provided herein are cells comprising any of the vectors provided herein. In some embodiments, the cells are immune cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are NK cells or T cells. In some embodiments, the cells are T cells. In some embodiments, the T cells are CD4+ T cells or CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In some embodiments, the T cells are primary T cells. In some embodiments, the cells are stem cells. In some embodiments, the stem cells are multipotent and pluripotent stem cells. In some embodiments, the stem cells are induced pluripotent stem cells (iPSCs). In some embodiments, the cells are differentiated from induced pluripotent stem cells. In some embodiments, the cells are allogeneic cells. In some embodiments, the cells are engineered to be hypoimmune.

[0050] In some embodiments, the cells exhibit cytotoxic activity against GPRC5D+ cells, BCMA+ cells, or GPRC5D+ / BCMA+ cells. In some embodiments, the cells exhibit cytotoxic activity against GPRC5D+ cells. In some embodiments, the cells exhibit cytotoxic activity against BCMA+ cells. In some embodiments, the cells exhibit cytotoxic activity against GPRC5D+ / BCMA+ cells. In some embodiments, the cells exhibit cytotoxic activity against GPRC5D+ cells, BCMA+ cells, and GPRC5D+ / BCMA+ cells.

[0051] Also provided herein is a composition comprising a plurality of the cells provided herein. In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0052] Also provided herein is a pharmaceutical composition comprising a plurality of any of the compounds provided herein and a pharmaceutically acceptable excipient.

[0053] In some embodiments, the composition comprises CD4+ T cells and CD8+ T cells. In some embodiments, the composition comprises a ratio of CD4+ T cells to CD8+ T cells of about 1:3 to about 3:1. In some embodiments, the composition comprises a ratio of CD4+ T cells to CD8+ T cells of about 1:2 to about 2:1. In some embodiments, the composition comprises a ratio of CD4+ T cells to CD8+ T cells of about 1:1.

[0054] In some embodiments, about 90% or more, about 95% or more, or about 99% or more of the cells in the composition are CD3+ T cells. In some embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells in the composition express a CAR. In some embodiments, of the plurality of cells in the composition that express a CAR, less than about 10%, about 9%, about 8%, about 7%, about 5%, about 4%, about 3%, about 2%, or about 1% of the cells exhibit sustained signaling.

[0055] In some embodiments, the composition comprises about 1.0 x 10 7 1.2 × 10 from CAR-expressing T cells 9 CAR-expressing T cells, approximately 1.0 × 10 7 6.5 × 10 from CAR-expressing T cells 8 CAR-expressing T cells, approximately 1.5 × 10 7 6.5 × 10 from CAR-expressing T cells 8 CAR-expressing T cells, approximately 1.5 × 10 7 6.0 × 10 from CAR-expressing T cells 8 CAR-expressing T cells, approximately 2.5 × 10 7 6.0 × 10 from CAR-expressing T cells 8 CAR-expressing T cells, approximately 5.0 × 10 7 6.0 × 10 from CAR-expressing T cells 8CAR-expressing T cells, approximately 1.25 × 10 7 1.2 × 10 from CAR-expressing T cells 9 CAR-expressing T cells, approximately 1.5 × 10 7 1.2 × 10 from CAR-expressing T cells 9 CAR-expressing T cells, approximately 5.0 × 10 7 4.5 × 10 from CAR-expressing T cells 8 CAR-expressing T cells, or approximately 1.5 × 10 8 3.0 × 10 from CAR-expressing T cells 8 In some embodiments, the composition comprises about 1.5 x 10 CAR-expressing T cells. 7 , about 2.5×10 7 , about 5.0×10 7 , about 7.5×10 7 , about 1.0×10 8 , about 1.25×10 8 , about 1.5×10 8 , about 1.75×10 8 , about 2×10 8 , about 2.25×10 8 , about 2.5×10 8 , about 3.0×10 8 , about 3.5×10 8 , about 4×10 8 , about 4.5×10 8 , about 6.0×10 8 , about 8.0×10 8 , or approximately 1.2 × 10 9 Includes CAR-expressing T cells.

[0056] Also provided herein are methods of treating a disease or condition, comprising administering to a subject any of the cells provided herein. In some embodiments, the cells are administered in a concentration of about 1 x 10 7 1 × 10 CAR-expressing T cells 9 In some embodiments, the CAR-expressing T cells are administered to the subject at a dose of up to about 2.5 x 10 7 Approximately 4.5 × 10 from CAR-expressing T cells 8 In some embodiments, the CAR-expressing T cells are administered to the subject at a dose of up to about 2.5 x 10 7The subject is administered a dose of CAR-expressing T cells. In some embodiments, the cells are administered at a dose of about 7.5 x 10 7 The subject is administered a dose of CAR-expressing T cells. In some embodiments, the cells are administered at a dose of about 1.5 x 10 8 The subject is administered a dose of CAR-expressing T cells. In some embodiments, the cells are administered at a dose of about 3.0 x 10 8 The subject is administered a dose of CAR-expressing T cells. In some embodiments, the cells are administered at a dose of about 4.5 x 10 8 A dose of CAR-expressing T cells is administered to the subject.

[0057] In some embodiments of claims 133-140, the method further comprises administering lymphocyte-depleting therapy to the subject prior to administration of the dose of CAR-expressing T cells. In some embodiments, the lymphocyte-depleting therapy is completed within about 7 days prior to initiating administration of the dose of CAR-expressing T cells. In some embodiments, administration of the lymphocyte-depleting therapy is completed within about 2 to 7 days prior to initiating administration of the dose of engineered T cells. In some embodiments, the lymphocyte-depleting therapy comprises administration of fludarabine and / or cyclophosphamide. In some embodiments, the lymphocyte-depleting therapy comprises administration of fludarabine and cyclophosphamide. In some embodiments, the lymphocyte-depleting therapy comprises administration of about 200-400 mg / m daily, inclusive of endpoints. 2 In some embodiments, the lymphocyte depletion therapy comprises administration of cyclophosphamide at a dose of about 300 mg / m daily. 2 In some embodiments, lymphocyte depletion therapy involves administration of cyclophosphamide at a dose of about 20-40 mg / m daily, inclusive of endpoints. 2 In some embodiments, the lymphocyte depletion therapy comprises administration of fludarabine at a dose of about 30 mg / m daily. 2 In some embodiments, the lymphocyte depletion therapy comprises administration of fludarabine and cyclophosphamide for 2 to 4 days. In some embodiments, the lymphocyte depletion therapy comprises administration of fludarabine and cyclophosphamide for 3 days.

[0058] In some embodiments, lymphocyte depleting therapy comprises administration of bendamustine. In some embodiments, lymphocyte depleting therapy comprises administration of about 50-130 mg / m daily, inclusive of endpoints. 2 In some embodiments, the lymphocyte depleting therapy comprises administration of bendamustine at a dose of about 90 mg / m daily. 2 In some embodiments, the lymphocyte depleting therapy comprises administration of bendamustine for 1 to 3 days. In some embodiments, the lymphocyte depleting therapy comprises administration of bendamustine for 2 days.

[0059] Also provided herein is the use of any of the cells provided herein in the manufacture of a medicament for treating a disease or condition in a subject. Also provided herein is the use of any of the cells provided herein for treating a disease or condition in a subject. Also provided herein is any of the cells provided herein for treating a disease or condition in a subject.

[0060] Also provided herein is a method for treating a disease or condition, comprising administering any of the compositions provided herein to a subject. Also provided herein is the use of any of the compositions provided herein in the manufacture of a medicament for treating a disease or condition in a subject. Also provided herein is the use of any of the compositions provided herein for treating a disease or condition in a subject. Also provided herein is any of the compositions provided herein for treating a disease or condition in a subject.

[0061] In some embodiments, the disease or condition is cancer. In some embodiments, the disease or condition is a plasma cell malignancy. In some embodiments, the disease or condition is a BCMA-expressing cancer and / or a GPRC5D-expressing cancer. In some embodiments, the disease or condition is a BCMA-expressing cancer. In some embodiments, the disease or condition is a GPRC5D-expressing cancer. In some embodiments, the disease or condition is a BCMA-expressing cancer and a GPRC5D-expressing cancer. In some embodiments, the disease or condition is multiple myeloma. In some embodiments, the disease or condition is relapsed / refractory multiple myeloma.

[0062] In some embodiments, the subject has received one or more prior therapies. In some embodiments, the subject has received at least one, but no more than three, prior therapies. In some embodiments, the prior treatment is a proteasome inhibitor, an immunomodulatory agent, an anti-CD38 antibody, prior treatment involved in autologous hematopoietic stem cell transplantation (HSCT), or any combination thereof. In some embodiments, the cells or compositions may be used in the manufacture of a medicament for treating a disease or condition in a subject. In some embodiments, the cells or compositions may be used to treat a disease or condition in a subject. In some embodiments, the disease or condition is cancer, optionally a plasma cell malignancy. In some embodiments, the disease or condition is a BCMA-expressing cancer and / or a GPRC5D-expressing cancer. In some embodiments, the disease or condition is multiple myeloma. In some embodiments, the disease or condition is relapsed / refractory multiple myeloma (RRMM).

[0063] In some embodiments, the subject has received one or more prior therapies. In some embodiments, the subject has received at least one, but not more than three, prior therapies. In some embodiments, the prior treatment is a proteasome inhibitor, an immunomodulatory agent, an anti-CD38 antibody, prior treatment involved in autologous hematopoietic stem cell transplantation (HSCT), or any combination thereof. In some embodiments, the cells or compositions are for treating a disease or condition in a subject. In some embodiments, the disease or condition is cancer, optionally a plasma cell malignancy. In some embodiments, the disease or condition is a BCMA-expressing cancer and / or a GPRC5D-expressing cancer. In some embodiments, the disease or condition is multiple myeloma. In some embodiments, the disease or condition is relapsed / refractory multiple myeloma (RRMM). In some embodiments, the subject has received one or more prior therapies. In some embodiments, the subject has received at least one, but not more than three, prior therapies. In some embodiments, the prior treatment is a proteasome inhibitor, an immunomodulatory agent, an anti-CD38 antibody, a prior treatment included in an autologous hematopoietic stem cell transplant (HSCT), or any combination thereof.

[0064] Also provided herein is a kit, comprising any of the CARs, polynucleotides, vectors, cells, or compositions provided herein and instructions for use.In some embodiments, the instructions are for administering the CARs, cells, or compositions.In some embodiments, the instructions specify that the CARs, cells, or compositions are administered to a subject with a disease or condition.

[0065] Also provided herein are articles of manufacture that include any of the CARs, polynucleotides, vectors, cells, compositions, or kits provided herein. [Brief explanation of the drawings]

[0066] [Figure 1-1]1A and 1B show the expression of human GPRC5D and human BCMA in various tumor cell lines as assessed by flow cytometry. [Figure 1-2] Same as above.

[0067] [Figure 2-1] Figure 2A shows the structure of an exemplary generated bispecific linear tandem CAR targeting GPRC5D and BCMA, with either the GPRC5D-binding domain (left panel) or the BCMA-binding domain (right panel) proximal to the cell membrane.

[0068] [Figure 2-2] Figure 2B shows the structures of exemplary generated bispecific loop tandem CARs targeting GPRC5D and BCMA, with either the GPRC5D-binding domain (left panel) or the BCMA-binding domain (right panel) proximal to the cell membrane.

[0069] [Figure 3-1] Figures 3A and 3B show antigen-independent (sustained) signaling (Figure 3A) and antigen-dependent signaling (Figure 3B), respectively, of Nurkat reporter cells expressing an exemplary generated bispecific tandem CAR, alone or after co-culture with target cells expressing GPRC5D and BCMA. [Figure 3-2] Same as above.

[0070] [Figure 4-1] Figures 4A and 4B show antigen-dependent activation of Nurkat reporter cells expressing an exemplary generated bispecific tandem CAR following single-antigen co-culture with MM.1S or OPM-2 cells knocked out for GPRC5D or BCMA, respectively. [Figure 4-2] Same as above.

[0071] [Figure 5]Figure 5 shows the percentage of cells that are surface positive for expression of each CAR, as assessed by flow cytometry (numbers indicate the top 14 tandem CAR constructs).

[0072] [Figure 6-1] Figure 6A shows the ability of T cells expressing the indicated tandem CAR constructs to lyse target cells after 21 days of co-culture (left to right: MM.1S, MM.1S BCMA KO, and MM.1S GPRC5D KO). *CAR T cells were unable to survive against MM.1S BCMA KO cells by day 21. ^CAR T cells were unable to survive against MM.1S GPRC5D KO cells by day 21.

[0073] [Figure 6-2] Figures 6B and 6C show the proliferation of CAR T cells expressing a linear tandem CAR construct (circle dots), a loop tandem CAR construct (circle dots), a single-targeting (GPRC5D or BCMA) CAR construct (square or diamond, respectively), or a bicistronic CAR construct (triangle) at days 7, 14, and 21 in coculture with MM.1S cells knocked out for BCMA (Figure 6B) or GPRC5D (Figure 6C). [Figure 6-3] Same as above.

[0074] [Figure 7-1] Figures 7A and 7B show separate plots of tumor burden over 49 days following treatment with a high dose (2 x 10) (Figure 7A) or a low dose (0.5 x 10) (Figure 7B) of T cells expressing the indicated CARs in the MM.1S mouse model of multiple myeloma. [Figure 7-2] Same as above.

[0075] [Figure 8]Figures 8A and 8B show the tumor control index (TCI) through 49 days after treatment with high (2x10) or low (0.5x10) doses, respectively, of T cells expressing the indicated CARs in the MM.1S mouse model of multiple myeloma.

[0076] [Figure 9-1] Figures 9A and 9B show separate plots of tumor burden over 49 days following treatment with a high dose (2 x 10) (Figure 9A) or a low dose (0.5 x 10) (Figure 9B) of T cells expressing the indicated CARs in the TPMI-8226 mouse model of multiple myeloma. [Figure 9-2] Same as above.

[0077] [Figure 10] Figure 10A and Figure 10B show the tumor control index (TCI) through 49 days after treatment with high (2 x 10) or low (0.5 x 10) doses, respectively, of T cells expressing the indicated CARs in the RPMI-8226 mouse model of multiple myeloma.

[0078] [Figure 11-1] Figure 11A shows individual plots of tumor burden over 28 days following treatment with 4 x 10 T cells expressing the indicated CARs (solid line) or mock-treated T cells (dashed line) in a mouse model of multiple myeloma antigenic heterogeneity.

[0079] [Figure 11-2] Figures 11B and 11C show the tumor control index (TCI) and tumor burden by bioluminescence imaging (BLI), respectively, through 28 days after treatment with 4 x 10 T cells expressing the indicated CARs in a mouse model of multiple myeloma antigenic heterogeneity. [Figure 11-3] Same as above.

[0080] [Figure 12-1]Figures 12A and 12B show the expression of both anti-GPRC5D scFv (y-axis) and anti-BCMA scFv (x-axis) in T cells from three human donors transduced with tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR, or mock-transduced cells. [Figure 12-2] Same as above.

[0081] [Figure 13-1] Figures 13A and 13B show the proliferation and CD25 expression of T cells transduced with bispecific tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR, or mock-transduced T cells, respectively, after co-culture with various cell lines. [Figure 13-2] Same as above.

[0082] [Figure 13-3] Figures 13C and 13D show the secretion of IFNγ (Figure 13C, upper panel), IL-2 (Figure 13C, lower panel), and TNFα (Figure 13D) by T cells transduced with bispecific tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR, or mock-transduced T cells after coculture with various cell lines. Graphs show the mean concentrations of pro-inflammatory cytokines, and data points represent cytokine levels for individual donors. [Figure 13-4] Same as above.

[0083] [Figure 14-1] Figure 14A shows CD25 expression by T cells transduced with bispecific tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR, or mock-transduced T cells after co-culture with various cell lines. Data points represent values from CAR T cells of individual donors.

[0084] [Figure 14-2]Figure 14B and Figure 14C show the secretion of IFNγ, IL-2, and TNFα (left, middle, or right panels, respectively) by T cells transduced with bispecific tandem CAR5 (Figure 14B), anti-BCMA CAR (Figure 14B), or anti-GPRC5D CAR (Figure 14C) after co-culture with various cell lines. Graphs show the mean concentrations of pro-inflammatory cytokines, and data points represent values from individual donors. [Figure 14-3] Same as above.

[0085] [Figure 15] Figure 15 shows the cytotoxic activity of CAR5, anti-BCMA CARs, and anti-GPRC5D CARs against tumor cell lines expressing various levels of BCMA and GPRC5D. Data are plotted as the mean and standard error across three donors.

[0086] [Figure 16-1] Figure 16A shows the number of CAR+ human CD3+ T cells per microliter of peripheral blood in MM.1S xenografted mice treated with 5x105 (low dose; left panel) or 2x106 (high dose; right panel) CAR T cells.

[0087] [Figure 16-2] Figure 16B (upper panel) shows the mean tumor volume for groups of MM.1S xenografted mice treated with 5x105 (low dose; left panel) or 2x106 (high dose; right panel) bispecific tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR transduced T cells, or mock-transduced T cells. Figure 16B (lower panel) shows individual tumor volumes for MM.1S xenografted mice treated with 5x105 (low dose; left panel) or 2x106 (high dose; right panel) anti-GPRC5D CAR T cells or mock-transduced T cells.

[0088] [Figure 16-3]Figure 16C (upper panel) shows individual tumor volumes for MM.1S xenografted mice treated with 5x105 (low dose; left panel) or 2x106 (high dose; right panel) anti-BCMA CAR T cells or mock-transduced T cells. Figure 16C (lower panel) shows individual tumor volumes for MM.1S xenografted mice treated with 5x105 (low dose; left panel) or 2x106 (high dose; right panel) bispecific tandem CAR5 T cells or mock-transduced T cells.

[0089] [Figure 16-4] Figure 16D shows the tumor control index for MM.1S xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR T cells, or mock-transduced T cells.

[0090] [Figure 16-5] Figure 16E shows the survival probability for MM.1S xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR T cells, or mock-transduced T cells.

[0091] [Figure 17-1] Figure 17A (upper panel) shows the average tumor burden for groups of OPM-2 xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR T cells, or mock-transduced T cells. Figure 17A (lower panel) shows the individual tumor burden for OPM-2 xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) anti-GPRC5D CAR T cells or mock-transduced T cells.

[0092] [Figure 17-2]Figure 17B (upper panel) shows the individual tumor burden for OPM-2 xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) anti-BCMA CAR T cells or mock-transduced T cells. Figure 17B (lower panel) shows the individual tumor burden for OPM-2 xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) tandem CAR5 T cells or mock-transduced T cells.

[0093] [Figure 17-3] Figure 17C shows the tumor control index for OPM-2 xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR T cells, or mock-transduced T cells.

[0094] [Figure 17-4] Figure 17D shows survival rates for OPM-2 xenografted mice treated with 5x105 (low dose) or 2x106 (high dose) tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR T cells, or mock-transduced T cells. DETAILED DESCRIPTION OF THE INVENTION

[0095] Detailed Description Provided herein are bispecific chimeric antigen receptors (CARs) (also referred to as "dual-targeting" CARs) that target or are directed to G protein-coupled receptor class C group 5 member D (GPRC5D) and B cell maturation antigen (BCMA). In some embodiments, the provided bispecific CARs target or are directed to GPRC5D-expressing and / or BCMA-expressing cells and diseases. Also provided are cells, such as T cells, engineered to express the provided bispecific CARs, and compositions containing such cells. GPRC5D is observed to be expressed, e.g., heterogeneously expressed, in certain diseases and conditions, such as malignant tumors, or in tissues or cells, e.g., malignant tumor plasma cells, such as those in patients with relapsed or newly diagnosed myeloma, while its expression in, e.g., normal tissues, remains low. Provided embodiments include approaches useful for treating diseases and conditions and / or targeting such cell types, including nucleic acid molecules encoding GPRC5D-binding and BCMA-binding domains, including chimeric antigen receptors (CARs), and the encoded receptors, such as the encoded CARs, as well as compositions and articles of manufacture comprising them. These receptors may generally contain antibodies specific for GPRC5D and BCMA (including antigen-binding antibody fragments, such as heavy chain variable (VH) regions, single-domain antibody fragments, and single-chain fragments, including scFvs). Also provided are cells, such as engineered or recombinant cells, that express such GPRC5D- or BCMA-binding receptors, e.g., bispecific CARs, and / or contain nucleic acids encoding such receptors, as well as compositions and articles of manufacture and therapeutic doses containing such cells.

[0096] The provided embodiment relates to CAR T cells targeting both GPRC5D and BCMA for the treatment of multiple myeloma. GPRC5D (Uniprot Acc. No. Q9NZD1, e.g., SEQ ID NO: 59) is a G protein-coupled receptor class C group 5 member D belonging to the RAIG (retinoic acid-inducible gene 1) family. It is a seven-transmembrane helix 39 kDa G protein-coupled receptor with two reported isoforms, the difference between which resides in the intracellular C-terminus of the protein. The results herein demonstrate that GPRC5D is expressed at high levels in multiple myeloma and at low levels overall in most normal tissues. BCMA (Uniprot Acc. No. Q02223, e.g., SEQ ID NO: 60) is a type III transmembrane protein expressed on mature B lymphocytes. After BCMA binds to its ligands, B-cell-activating factor of the TNF family (BAFF) or proliferation-inducing ligand (APRIL), it transmits a pro-survival signal to B cells, which is known to be required for plasma cell survival.

[0097] Multiple myeloma (MM) is a hematological malignancy characterized by the uncontrolled proliferation of monoclonal plasma cells in the bone marrow, resulting in the overproduction of monoclonal immunoglobulins and immunosuppression (Al-Hujaily 2016; Dimopoulos 2015). Adoptive T cell therapies, such as CAR T cell therapy, have shown promise in the treatment of multiple myeloma, with clinical efforts primarily targeting the B cell maturation antigen (BCMA). Indeed, recent advances in MM treatment options have included FDA approval of two chimeric antigen receptor (CAR) T cell therapies targeting BCMA. However, although BCMA is expressed on many malignant plasma cells, expression levels can be heterogeneous in some cases. In some aspects, heterogeneity in target antigen expression can lead to variable or inconsistent responses. In some aspects, cell surface BCMA expression has also been observed to change over time due to gamma-secretase-mediated shedding of the extracellular domain. While multiple clinical trials have demonstrated high overall response rates, most patients ultimately relapse, and decreased BCMA expression has been observed after CAR T-cell therapy (Brudno et al. (2018) J. Clin. Oncol., JCO2018778084, Cohen et al. (2017) Blood 130:505). Targeting secondary antigens in MM may overcome antigen downregulation or loss, thereby reducing the chance of immune evasion. For example, both BCMA and GPRC5D are highly expressed in MM, but their expression is independent of the other, making them a promising combination for dual targeting (Smith et al., Sci Transl Med (2019) 11(485):aau7746). Notably, the CARs provided herein do not demonstrate appreciable recombination (e.g., homologous recombination).In contrast, dual-targeting CARs, formatted in a bicistronic arrangement to allow expression of two independent CARs from a single vector, may exhibit unexpected or unwanted recombination due to high sequence homology between different portions of the vector (e.g., portions encoding identical or similar components of each independent CAR). Lam et al., Blood (2021) 138 (Suppl. 1):4808.

[0098] Also, in some contexts, recombinant receptors may exhibit antigen-independent activity or signaling (also known as "sustained signaling"), which may lead to undesirable effects, such as due to increased differentiation and / or exhaustion of T cells expressing the recombinant receptor. In some embodiments, such activity may limit the activity, efficacy, or potency of T cells. In some cases, during engineering and ex vivo expansion of cells to express the recombinant receptor, the cells may exhibit a phenotype suggestive of exhaustion due to sustained signaling through the recombinant receptor. In some cases, alternative or additional T cell therapy approaches targeting MM are needed.

[0099] The engineered cells provided include cells containing a chimeric antigen receptor that exhibits high expression of both the BCMA-binding domain and the GPRC5D-binding domain, and low persistent signaling, thereby minimizing the possibility of antigen-independent (persistent) signaling. In particular, the bispecific CARs provided herein include CARs with high antigen-dependent activation and minimal persistent signaling.

[0100] Provided is a monotherapy approach that uses bispecific CAR that targets both GPRC5D and BCMA expressed in autologous primary T cells as a therapeutic agent for the plasma cells of multiple myeloma.In some embodiments, monotherapy may be desirable for subjects that are known to have, suspected to have, or selected to have low or heterogeneous MM plasma cells with BCMA expression.GPRC5D and BCMA have been observed to be expressed, for example, heterogeneously expressed, in certain diseases or conditions, such as malignant tumors, or in tissues or cells, for example, malignant tumor plasma cells from recurrent or newly diagnosed myeloma patients, whose expression is slight or low in normal tissues.Due to the role of GPRC5D and BCMA in various diseases and conditions, including cancer, both GPRC5D and BCMA are therapeutic targets.

[0101] In some cases, simultaneous targeting of both antigens provided herein may improve the depth and durability of responses across patients, in addition to minimizing relapse due to antigen escape. The mechanism of resistance to CAR T cell therapy may be loss or downregulation ("escape") of the target antigen, as supported by data from CAR T cell clinical trials in B-cell malignancies. (Robbie G. Majzner and Crystal L. Mackall, Cancer Discov August 22, 2018; DOI 10.1158 / 2159-8290.CD-18-0442). Such dual targeting strategies may achieve synergistic or improved tumor responses based on targeting two antigens compared to approaches involving only single antigen targeting. A dual targeting approach may be advantageous in MM to overcome challenges due to potential antigen loss and / or maximize antigen targeting.

[0102] Furthermore, the CARs provided herein exhibit strong in vitro function against three different multiple myeloma cell lines and strong in vivo efficacy across three different multiple myeloma models, supporting their suitability in the presence of various antigen levels, up to complete antigen loss. To this end, the observations herein demonstrate that the provided CARs are highly functional when signaling through a single binding domain, which is consistent with the observation that the CARs will exhibit anti-tumor efficacy in the presence of only a single antigen (i.e., GPRC5D or BCMA), for example, in the case of antigen loss.

[0103] Among the embodiments provided are approaches useful for treating diseases and conditions and / or targeting such cell types, including nucleic acid molecules encoding bispecific chimeric antigen receptors that bind both GPRC5D and BCMA, and the encoded receptors, such as encoded CARs, and compositions and articles of manufacture comprising same. The receptors are generally antibodies (heavy chain variable (V)) specific for GPRC5D and BCMA. H ) regions, single domain antibody fragments, and single chain fragments including short chain variable fragments (scFv). Also provided herein are cells, such as engineered or recombinant cells, that express such CARs and / or contain nucleic acids encoding such receptors, as well as compositions and articles of manufacture and therapeutic doses containing such cells.

[0104] All publications referenced in this application, including patent literature, scientific literature, and databases, are incorporated by reference in their entirety for all purposes to the same extent as if each publication were individually incorporated by reference. To the extent that a definition set forth herein contradicts or otherwise conflicts with a definition set forth in a patent, application, application publication, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0105] The section headings used herein are for formatting purposes only and are not to be construed as limiting the subject matter described.

[0106] I. Recombinant Receptors (e.g., Chimeric Antigen Receptors) In some embodiments, provided are GPRC5D- and BCMA-binding substances, such as recombinant receptors or chimeric antigen receptors (CARs), comprising extracellular binding domains that bind to both GPRC5D and BCMA. The extracellular binding domain comprises a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA. The GPRC5D-binding domain comprises a cell surface protein containing an antibody (e.g., an antigen-binding antibody fragment) and / or other binding peptide that specifically binds to GPRC5D (e.g., human GPRC5D protein). The BCMA-binding domain comprises a cell surface protein containing an antibody (e.g., an antigen-binding antibody fragment) and / or other binding peptide that specifically binds to BCMA (e.g., human BCMA). In some embodiments, the binding domain binds to the extracellular portion of GPRC5D. In some embodiments, the GPRC5D-binding domain binds to the extracellular portion of GPRC5D. In some embodiments, the binding domain binds to the extracellular portion of BCMA. In some embodiments, the BCMA-binding domain binds to the extracellular portion of BCMA.

[0107] Among the provided polynucleotides are those that encode recombinant receptors, such as antigen receptors, that specifically bind to GPRC5D and BCMA. In some embodiments, the encoded receptors, such as those containing GPRC5D- and BCMA-binding polypeptides, as well as compositions and articles of manufacture and uses thereof, are also provided.

[0108] The GPRC5D and BCMA binding domains include antibodies, such as single-chain antibodies (e.g., antigen-binding antibody fragments), or portions thereof. In some examples, the recombinant receptor contains a GPRC5D antibody or antigen-binding fragment thereof and an anti-BCMA antibody or antigen-binding fragment thereof, for example, in tandem. The provided polynucleotides can be incorporated into constructs, such as deoxyribonucleic acid (DNA) or RNA constructs, that can be introduced into cells for expression of the encoded recombinant GPRC5D binding domain and recombinant BCMA binding domain.

[0109] The provided recombinant receptors generally contain an extracellular binding domain and an intracellular signaling domain. The provided receptors include polypeptides containing antibodies, such as anti-GPRC5D antibodies and anti-BCMA antibodies. Such receptors include chimeric antigen receptors containing such antibodies.

[0110] The provided recombinant receptor has an extracellular binding domain including a GPRC5D-binding domain and a BCMA-binding domain. The recombinant receptor includes a GPRC5D-binding domain that specifically binds to GPRC5D, such as an anti-GPRC5D antibody, e.g., a GPRC5D antigen-binding fragment. The recombinant receptor also includes a BCMA-binding domain that specifically binds to BCMA, such as an anti-BCMA antibody, e.g., a BCMA antigen-binding fragment. The antigen receptor includes a functional non-TCR antigen receptor, such as a chimeric antigen receptor (CAR). Also provided are cells expressing the recombinant receptor and their use in adoptive cell therapy, such as the treatment of diseases or disorders associated with GPRC5D expression, BCMA expression, or both, e.g., multiple myeloma.

[0111] Chimeric receptors include chimeric antigen receptors (CARs).CARs generally comprise an extracellular binding domain, including a GPRC5D binding domain and a BCMA binding domain, a transmembrane domain, and an intracellular signaling domain.CARs generally also comprise a spacer sequence (such as a hinge sequence) between the extracellular binding domain and the transmembrane domain.The exemplary features of the provided CARs are described in the following subsections.

[0112] 1. Extracellular antigen-binding domain A chimeric receptor such as a CAR generally comprises an extracellular binding domain that comprises, is, or comprises an anti-GPRC5D antibody and an anti-BCMA antibody. Thus, a chimeric receptor, e.g., a CAR, typically comprises a GPRC5D-binding domain and a BCMA-binding domain in its extracellular portion, such as an antigen-binding fragment, domain, or portion, or one or more antibody variable regions, and / or antibody molecules, such as those described herein.

[0113] In some embodiments, the extracellular antigen-binding domain comprises a GPRC5D-binding domain and a BCMA-binding domain. In some embodiments, the GPRC5D-binding domain comprises an anti-GPRC5D antibody or an antigen-binding fragment thereof. In some embodiments, the BCMA-binding domain comprises an anti-BCMA antibody or an antigen-binding fragment thereof.

[0114] As used herein, the term "antibody" is used in the broadest sense and includes intact antibodies as well as fragments antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) fragments capable of specifically binding to an antigen. H (scFv) regions, single-chain antibody fragments including single-chain variable fragments (sdAb, sdFv, nanobody) fragments, and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific or trispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFvs, tandem tri-scFvs, and the like. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof, also referred to herein as "antigen-binding fragments." The term also encompasses intact or full-length antibodies, which include antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0115] "Complementarity-determining region" and "CDR," synonyms of "hypervariable region" or "HVR," are known to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, and CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, and CDR-L3) in each light chain variable region. "Framework region" and "FR" are known to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0116] The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering system); Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48 ("Chothia" numbering system); MacCallum et al., J. Mol. Biol, 1996; 262:732-745 ("Contact" numbering system); Lefranc MP et al., Dev Comp Immunol, 2003; 27(1):55-77 ("IMGT" numbering system); Honegger A and Pluckthun A, J Mol Biol, 2001; 309(3):657-70 ("Aho" numbering system); Martin et al., PNAS, 1989; 86(23):9268-9272 ("AbM" numbering system); and Ye et al., Nucleic Acids Res. 2013; 41 (web server article):W34-40 ("IgBLAST" numbering system).Details regarding various numbering systems are also provided, for example, in Jarasch et al., Proteins, 2017; 85(1):65-71; Martin et al., Bioinformatics tools for antibody engineering; Dubel, S. (editor) Handbook of Therapeutic Antibodies, Vol. 1. Wiley-VCH, Weinheim, Germany; Martin, ACR (2010). Protein Sequence and Structure Analysis of Antibody Variable Domains; Kontermann, R., Dubel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg; and Martin, ACR, Antibody Information: How to identify the CDRs by looking at a sequence [online] bioinf.org.uk / abs / info.html, all of which are incorporated herein by reference in their entireties. A variety of predictive algorithm tools for numbering antibody residues and CDRs are available and known (e.g., AbYsis, Abnum, AbYmod, AbRSA, IgBLAST, IMGT, or ANARCI).

[0117] The boundaries of a given CDR or FR may vary depending on the system used for identification. For example, the Kabat system is based on structural alignment, while the Chothia system is based on structural information. Both the Kabat and Chothia numbering systems are based on the most common antibody region sequence lengths, with insertions in some cases. Insertions in a sequence relative to the standard numbering system are indicated using an insertion letter code. For example, residues inserted between residues L30 and L31 are indicated as L31A, L31B, etc. Deletions in a sequence relative to the standard system are accommodated by skipping a number. These two systems place certain insertions and deletions ("indels") in different positions, resulting in different numbering. For example, the Chothia numbering system is nearly identical to the Kabat numbering system, except that insertions are placed based on structural position, and topologically equivalent residues are assigned the same number. The Contact system is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering system. The AbM scheme is a compromise of the Kabat and Chothia definitions and is based on that used by Oxford Molecular's AbM antibody modeling software. The IgBLAST scheme is based on matching to germline V, D, and J genes and can be determined using the IgBLAST tool from the National Center for Biotechnology Information (NCBI).

[0118] In some embodiments, Kabat numbering can be determined by known sequence rules, e.g., as described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.In some embodiments, the Kabat numbering system may, in some aspects, include any of the following rules for naming the CDRs: CDR-L1 begins at approximately residue 24 of the light chain and always has a preceding C residue and always has a following W residue; the end of CDR-L1 is defined by a stretch of three residues, where the W residue may be followed by Y, L, or F, which may be followed by Q or L; CDR-1 has a length of 10 to 17 residues; CDR-L2 always begins 16 residues after the end of CDR-L1; CDR-L2 The two residues preceding the CDR-L1 are I and Y, but can also be V and Y, I and K, or I and F; CDR-L2 is always 7 residues in length; CDR-L3 always starts 33 residues after the end of CDR-L2, always has a preceding C residue, and is followed strictly by an FGXG sequence motif, where X is any amino acid; CDR-L3 has a length of 7 to 11 residues; CDR-H1 starts at approximately position 26 of the heavy chain; the first amino acid of CDR-H1 is always 9 residues after the conserved C residue; CDR-H1 contains an invariant W residue CDR-H1 is followed by a group, typically followed by V, but can also be I or A; CDR-H1 has a length of 5 to 7 residues; CDR-H2 always begins 15 residues after the end of CDR-H1; the first residue of CDR-H2 is usually preceded by the sequence motif LEWIG, although some variation exists; the end of CDR-H2 is defined by a three-residue motif—the first residue of the three-residue motif can be either K or R, the second residue of the three-residue motif can be L, I, V, F, T, or A, and the last residue of the three-residue motif can be L, I, V, F, T, or A. the third residue can be T, S, I, or A; CDR-H2 has a length of 16 to 19 residues; CDR-H3 always starts 33 residues after the end of CDR-H2 and is always 3 residues after the C residue - the first residue of CDR-H3 is preceded by a conserved C residue, which is usually followed by two residues that are AR; residues following CDR-H3 are strictly followed by a WGXG sequence motif, where X is any amino acid; CDR-H3 typically has a length of 3 to 25 residues; CDR-H3 can be longer than 25 residues.

[0119] In some cases, the exact boundary position of a particular CDR according to the Chothia numbering system may vary based on different definitions of CDRs (see, e.g., Martin, ACR, Antibody Information: How to identify the CDRs by looking at a sequence [online] bioinf.org.uk / abs / info.html). For example, in some cases, the CDR-L1 boundary position according to Chothia numbering may be L26-L32 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk AM J Mol Biol, 1987; 196(4):901-17). In some cases, the CDR-L1 boundary position may be L25-L32 (Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, the boundary positions for CDR-L2 may be L50-L52, and for CDR-L3 may be L91-L96 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk AM J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, the boundary position of CDR-H1 according to Chothia numbering may be H26-H32 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk AM J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48).In some cases, the CDR-H2 boundary position can be H53-H55 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk AM J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48); H52a-H55 (Tramontano et al., J Mol Biol, 1990, 215(1): 175-82); or H52-H56 (Al-Lazikani et al., J Mol Biol., 1997; 273(4):927-48). In some cases, the CDR-H3 boundary position may be H96-H101 (Chothia et al., Science, 1986; 233(4765):755-8 and Chothia C. and Lesk AM J Mol Biol., 1987; 196(4):901-17). In some cases, the CDR-H3 boundary position may be H92-H104 (Morea et al., Biophys Chem, 1997; 68(1-3):9-16 and Morea et al., J Mol Biol., 1998; 275(2):269-94).

[0120] Table 1 below illustrates exemplary numbering and lists exemplary boundary positions for CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3, as identified by the Kabat, Chothia, AbM, and Contact systems, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering systems. FRs are located between the CDRs. For example, FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, etc. Note that because the Kabat numbering system shown places insertions at H35A and H35B, when numbered using the Kabat numbering system shown, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop. [Table 1]

[0121] Thus, unless otherwise specified, the "CDR" or "complementarity determining region" of a given antibody or region thereof, such as a variable region thereof, or each designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass the complementarity determining region (or the specific complementarity determining region) defined by any of the methods mentioned above or other known methods. For example, if a particular CDR (e.g., CDR-H3) is a CDR of a given V H or V L When a variable region amino acid sequence is described as containing the amino acid sequence of a corresponding CDR, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region defined in any of the ways mentioned above or otherwise known. In some embodiments, an antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR-H3 in a given V region. Hand CDR-L1, CDR-L2, and CDR-L3 are contained in a predetermined V region amino acid sequence. L The CDRs are defined by any of the previously mentioned methods, such as Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods, or other known methods. In some embodiments, specific CDR sequences are specified. While exemplary CDR sequences of the provided antibodies are described using various numbering systems, it is understood that the provided antibodies can contain CDRs described according to any of the previously mentioned numbering systems or other known numbering systems.

[0122] Similarly, unless otherwise specified, a FR or an individual designated FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and / or FR-L4) of a given antibody or region thereof, such as a variable region, should be understood to encompass a framework region (or said specific framework region) defined by any known method. In some cases, a method for identifying a particular CDR, FR, or FR(s) or CDR(s) is specified, such as "CDRs defined by Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods, or other known methods." In other cases, the specific amino acid sequence of a CDR or FR is provided. In some embodiments, an antibody or antigen-binding fragment thereof may incorporate FR-H1, FR-H2, FR-H3, and FR-H4 into a given V H The amino acid sequence of the region FR-L1, FR-L2, FR-L3, and FR-L4 is contained within the region FR-L1, FR-L2, FR-L3, and FR-L4. L When a region is described as being contained within an amino acid sequence, the FR may be defined by any of the methods mentioned above, such as Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods, or by other known methods.

[0123] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy and light chains (V, respectively) of a native antibody H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007)). A single V H or V L Furthermore, an antibody that binds to a specific antigen may have a V domain that is sufficient for binding to that antigen. H or V L domains to form complementary V L or V H They may also be isolated by screening libraries of domains (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0124] The antibodies provided include antibody fragments. An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of the intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab)'2, diabodies; linear antibodies; heavy chain variable (V) antibodies; H ) region, single-chain antibody molecules such as scFv and V H In some embodiments, the antibody comprises a variable heavy chain (VH), ... H ) and variable light chain (V L In certain embodiments, the antibody is or comprises an antibody fragment comprising a heavy chain variable (V) region, such as an scFv. H) region and / or light chain variable (V L ) region.

[0125] Single domain antibodies (sdAbs) are antibody fragments that contain all or part of the heavy chain variable region or all or part of the light chain variable region of an antibody. In certain embodiments, single domain antibodies are human single domain antibodies.

[0126] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolysis of intact antibodies and production by recombinant host cells. In some embodiments, antibodies are recombinantly produced fragments, such as fragments containing non-naturally occurring arrangements, such as those in which two or more antibody regions or chains are linked by synthetic linkers, e.g., peptide linkers, and / or fragments that may not be produced by enzymatic digestion of a native intact antibody. In some embodiments, the antibody fragment is an scFv.

[0127] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has been humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with the corresponding residues of a non-human antibody (e.g., the antibody from which the CDR residues were derived), e.g., to restore or improve antibody specificity or affinity.

[0128] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or other non-human source utilizing human antibody-encoding sequences, including a human antibody repertoire or human antibody library. The term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term includes antigen-binding fragments of human antibodies.

[0129] Human antibodies may be prepared by administering an immunogen to transgenic animals engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which replace endogenous immunoglobulin loci or are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies may also be derived from human antibody libraries, including phage display and cell-free libraries, containing antibody coding sequences from the human repertoire.

[0130] The provided antibodies include monoclonal antibodies, including monoclonal antibody fragments. As used herein, the term "monoclonal antibody" refers to substantially homogeneous antibodies, i.e., antibodies obtained from or within a population in which each individual antibody is identical except for variants containing naturally occurring mutations or variants that may arise during monoclonal antibody preparation (such variants are generally less abundant). In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the antigen. This term is not intended to require production of the antibody by any particular method. Monoclonal antibodies may be produced by a variety of techniques, including, but not limited to, hybridomas, recombinant DNA techniques, phage display, and other antibody display methods.

[0131] In some embodiments, the GPRC5D-binding domain comprises a heavy chain variable (V H ) region and the light chain variable (V L In some embodiments, the BCMA binding domain comprises a heavy chain variable (V H ) region and the light chain variable (V L ) area.

[0132] In some embodiments, the extracellular binding domain comprises a loop format. In some embodiments, from N-terminus to C-terminus, the extracellular domain comprises: a V of the BCMA binding domain; H Area and V L One of the regions; V of the GPRC5D binding domain H Area and V L One of the regions; V of the GPRC5D binding domain H Area and V L the other of the regions; and V of the BCMA binding domain H Area and V L The other side of the realm.

[0133] In some embodiments, the extracellular binding domain comprises a loop format. In some embodiments, from N-terminus to C-terminus, the extracellular domain comprises: the V of the GPRC5D binding domain H Area and V L One of the regions; V of the BCMA binding domain H Area and V L One of the regions; V of the BCMA binding domain H Area and V L the other of the regions; and V of the GPRC5D-binding domain H Area and V L The other side of the realm.

[0134] In some embodiments, the extracellular binding domain comprises a linear format. In some embodiments, from N-terminus to C-terminus, the extracellular domain comprises: the V of the GPRC5D binding domain H Area and V L One of the regions; V of the GPRC5D binding domain H Area and V L The other region: V of the BCMA binding domain H Area and V L one of the regions; and V of the BCMA binding domain H Area and V L The other side of the realm.

[0135] In some embodiments, the extracellular binding domain comprises a linear format. In some embodiments, from N-terminus to C-terminus, the extracellular domain comprises: V of the BCMA binding domain; H Area and V L One of the regions; V of the BCMA binding domain H Area and V L The other region: V of the GPRC5D binding domain H Area and V L one of the regions; and V of the GPRC5D-binding domain H Area and V L The other side of the realm.

[0136] a. GPRC5D binding domain In some embodiments, the provided GPRC5D-binding domain of the provided CAR contains an antibody, such as an anti-GPRC5D antibody, or an antigen-binding fragment thereof, that confers the GPRC5D-binding ability of the provided CAR. In some embodiments, the CAR comprises a heavy chain variable (V) of the antibody. H ) region and / or light chain variable (V L In some embodiments, the antibody comprises a GPRC5D-binding domain, such as the (V H ) area and (V L ) region is part of a tandem dual-targeting CAR with a BCMA binding domain. In some embodiments, the (V H ) area and (V L ) regions of the GPRC5D-binding domain are connected by a linker. H ) area and (V L ) region comprises an scFv antibody fragment. In some embodiments, the antibody or antigen-binding domain can be any of the GPRC5D antibodies described or can be derived from any of the anti-GPRC5D antibodies described (see, e.g., WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245). Any of these anti-GPRC5D antibodies or antigen-binding fragments can be used in the provided CARs. In some embodiments, the CAR comprises a heavy chain variable (V) derived from an antibody described in WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, or WO2018147245. H ) and / or light chain variable (V L ) region.

[0137] In some cases, the antibody, e.g., anti-GPRC5D antibody, or antigen-binding fragment, comprises a heavy and / or light chain variable (V H or V L ) region sequences, or a sufficient antigen-binding portion thereof. In some embodiments, an anti-GPRC5D antibody, e.g., an antigen-binding fragment, comprises a V nucleotide sequence containing the described CDR-H1, CDR-H2, and / or CDR-H3. H In some embodiments, an anti-GPRC5D antibody, e.g., an antigen-binding fragment, comprises a V domain sequence containing the CDR-L1, CDR-L2, and / or CDR-L3 described herein. L In some embodiments, the anti-GPRC5D antibody, e.g., antigen-binding fragment, contains a V domain sequence containing the described CDR-H1, CDR-H2, and / or CDR-H3. H V containing the region sequence and containing the CDR-L1, CDR-L2, and / or CDR-L3 described L Also included are antibodies having a sequence that is at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to such a sequence.

[0138] In some cases, the antibody or antibody fragment in the provided CAR comprises any V of the antibodies or antibody-binding fragments described in any of WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245. H It has an area.

[0139] In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 7, or the heavy chain variable (V H) region amino acids having an amino acid sequence that has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the V H V having a region or such H The antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and / or CDR-H3 appearing in sequence.

[0140] In some embodiments, the V of the antibody or antigen-binding fragment thereof H The regions include CDR-H1, CDR-H2, and / or CDR-H3 according to Kabat numbering. H The regions include CDR-H1, CDR-H2, and / or CDR-H3 according to Chothia numbering. H The regions include CDR-H1, CDR-H2, and / or CDR-H3 according to AbM numbering.

[0141] In some embodiments, the CAR comprises a heavy chain variable (V) comprising a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 3. H ) region.

[0142] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively. H Includes the area.

[0143] In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NOs: 1, 2, and 3. H Includes the area.

[0144] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the V and V sequences shown in SEQ ID NO: 7, respectively. H The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the region amino acid sequence.

[0145] In some embodiments of the antibodies or antigen-binding fragments thereof provided herein, V H The region includes any of the CDR-H1, CDR-H2, and CDR-H3 described above, and each of the V and V regions shown in SEQ ID NO: 7. H The region comprises framework region 1 (FR1), FR2, FR3, and / or FR4 having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to FR1, FR2, FR3, and / or FR4 contained within the amino acid sequence.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof comprises a V H Includes the area.

[0147] In some embodiments, V H The antibody or antibody fragment in the provided CAR comprising the region further comprises a light chain or a sufficient antigen-binding portion thereof. For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises a V H Area and V L Area, or V H Area and V L In some embodiments, the V H The region sequence is V H In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.

[0148] In some embodiments, the CARs provided herein are selected from the group consisting of the V H In some embodiments, the CAR comprises an antibody, such as an anti-GPRC5D antibody, containing either the V or VIII region and a light chain variable region or a sufficient antigen-binding portion thereof. H Region and light chain variable (V L ) area, or V H and V L In some embodiments, the V H The region sequence is V H In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.

[0149] In some embodiments, the antibody or antigen-binding fragment is selected from the group consisting of antibodies, polypeptides, and polypeptides described in any of WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245. L It has an area.

[0150] In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 8, or the light chain variable (V) L ) region amino acids having an amino acid sequence that has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the V L V having a region or such L The antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, and / or CDR-L3 appearing in sequence.

[0151] In some embodiments, the V of the antibody or antigen-binding fragment thereof L The V region comprises CDR-L1, CDR-L2, and CDR-L3 according to Kabat numbering. L The V region comprises CDR-L1, CDR-L2, and CDR-L3 according to Chothia numbering. L The regions include CDR-L1, CDR-L2, and / or CDR-L3 according to AbM numbering.

[0152] In some embodiments, the CAR comprises a light chain variable (V) comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 6. L ) region.

[0153] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-L1, a CDR-L2, and a CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. L Includes the area.

[0154] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the V and V sequences set forth in SEQ ID NO: 8, respectively. L The region contains CDR-L1, CDR-L2, and CDR-L3 contained within the amino acid sequence.

[0155] The CARs provided herein include those in which the antibody, such as an anti-GPRC5D antibody, or antibody fragment in the provided CAR has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7. Ha V region amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; L There is a CAR, which contains a region amino acid sequence.

[0156] In some embodiments, the V of the antibody or antigen-binding fragment thereof H The regions are V and V shown in SEQ ID NO: 7, respectively. H CDR-H1, CDR-H2, and CDR-H3 each having the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the amino acid sequence of the V region; L The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the amino acid sequence of the CDR-L1, CDR-L2, and CDR-L3 regions.

[0157] In some embodiments, the V of the antibody or antigen-binding fragment thereof H The V region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 7. L The V region comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the V region of the antibody or antigen-binding fragment thereof H and V L The regions are the amino acid sequences shown in SEQ ID NOs: 7 and 8, respectively, or the V H and V L The present invention also includes any antibody or antigen-binding fragment thereof having at least 90% sequence identity, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, to any of the following:

[0158] For example, the V of the antibodies or antigen-binding fragments provided herein H and V L The regions comprise the amino acid sequences shown in SEQ ID NOs: 7 and 8, respectively.

[0159] The provided CARs include those in which the GPRC5D-binding domain comprises a V CAR comprising the sequence set forth in SEQ ID NO:7, or an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:7. H and comprising the sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to SEQ ID NO:8. L In some embodiments, the GPRC5D-binding domain of the provided CAR comprises a V region having CDRH1, CDRH2, and CDRH3 containing the amino acid sequences of SEQ ID NOs: 1, 2, and 3, respectively. H V region, and CDRL1, CDRL2, and CDRL3 containing the amino acid sequences of SEQ ID NOs: 4, 5, and 6, respectively. L In some embodiments, the V H The region comprises the amino acid sequence set forth in SEQ ID NO: 7, and L The region comprises the amino acid sequence shown in SEQ ID NO:8.

[0160] In some embodiments, the GPRC5D-binding domain in the provided CAR is an antibody or antigen-binding fragment thereof, which is a single-chain antibody fragment such as a single-chain variable fragment (scFv) or diabody, or a single-domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is H In some embodiments, the antibody or antigen-binding fragment is a single domain antibody comprising only the heavy chain variable (V H ) region and the light chain variable (V L In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (V H ) region and the light chain variable (V L In some embodiments, a single-chain antibody fragment (e.g., an scFv) comprises a heavy chain variable (V H) region and the light chain variable (V L The linker comprises one or more linkers connecting two antibody domains or regions, such as the nucleotide sequence (SEQ ID NO: 1) and the nucleotide sequence (SEQ ID NO: 2). The linker is typically a peptide linker, e.g., a flexible and / or soluble peptide linker. The linker may be glycine- and serine-rich, and / or, in some cases, threonine-rich. In some embodiments, the linker further comprises charged residues, such as lysine and / or glutamic acid, which may improve solubility. In some embodiments, the linker further comprises one or more prolines.

[0161] Thus, in some embodiments, the CARs provided are typically V-type antibodies, such as scFvs and diabodies, particularly human single-chain antibody fragments. H and V L In some embodiments, the CARs provided contain anti-GPRC5D antibodies, including single-chain antibody fragments, that include a linker connecting two antibody domains or regions, such as V domains. ... H and V L The present invention also includes anti-BCMA antibodies, including single chain antibody fragments, that include a linker connecting two antibody domains or regions, such as a nucleotide sequence. The linker is typically a peptide linker, e.g., a flexible and / or soluble peptide linker, such as one rich in glycine and serine.

[0162] In some embodiments, the V of the GPRC5D-binding domain H and V L The region sequence is the V of at least one intervening BCMA binding domain. H and V L In some embodiments, the extracellular antigen-binding domain of the CAR is linked in sequence by a VH and VH region sequence of the GPRC5D binding domain as a loop CAR. L The VH and VH regions are from other BCMA binding domains. LIn some embodiments, the V of the GPRC5D-binding domain has a loop structure separated by one of the V regions. H or V L At least one of the domain sequences is connected to the V of the BCMA binding domain via a linker. H and V L Directly connected to the region.

[0163] In some embodiments, the CAR comprises a loop format. In some embodiments, the V of the BCMA binding domain H or V L The V region of the GPRC5D-binding domain is separated by a linker. H or V L In some embodiments, the VH and VH regions of the BCMA binding domain are linked to L One of the regions contains the VH and VH domains of the BCMA binding domain. L In some embodiments, the V of the GPRC5D-binding domain is linked to the other of the V H and V L One of the regions is connected to the V of the GPRC5D binding domain by a linker. H and V L In some embodiments, the linker is represented by SEQ ID NO: 17. In some embodiments, the linker is represented by SEQ ID NO: 18. In some embodiments, the linker is represented by SEQ ID NO: 19. In some embodiments, the linker is represented by SEQ ID NO: 21. In some embodiments, the linker is represented by SEQ ID NO: 22.

[0164] In some embodiments, the V of the BCMA binding domain H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. L In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. L In some embodiments, the V of the BCMA binding domain is linked to HThe region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. L In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. H In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. H In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. H is connected to the region.

[0165] In some embodiments, the V of the BCMA binding domain L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. L In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. L In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. L In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. H In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. H In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. H is connected to the region.

[0166] In some embodiments, the extracellular binding domain of the CAR comprises the V of the GPRC5D binding domain. H and V L The domains are directly linked by a linker in sequence (e.g., as an scFv), and the V of the BCMA binding domain H and V L In some embodiments, the GPRC5D binding domain has a linear format in which the domains are directly linked by a sequential linker (e.g., as an scFv). H Area and V L In some embodiments, the GPRC5D-binding domain comprises, in order from N- to C-terminus, V H and V L One of the regions, the linker, and V H and V L In some embodiments, the linker is represented by SEQ ID NO: 17. Thus, in some embodiments, the GPRC5D-binding domain comprises, from N to C terminus, V H and V L One of the regions, a linker shown in SEQ ID NO: 17, and V H and V L The other of the regions.

[0167] In some embodiments, glycine and serine (and / or threonine) rich linkers comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, they comprise at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and / or threonine. In some embodiments, the linker comprises substantially entirely glycine, serine, and / or threonine. Linkers are generally about 5 to about 50 amino acids in length, typically about 10 to about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples, 10 to 25 amino acids in length. Exemplary linkers include linkers having multiple repeats of the sequence GGGGS (4GS; SEQ ID NO:21) or GGGS (3GS; SEQ ID NO:20), e.g., 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of the sequences set forth in SEQ ID NO:22 (GGGGSGGGGS), SEQ ID NO:23 (GGGGSGGGGSGGGGS), and SEQ ID NO:24 (GGGGSGGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequences set forth in SEQ ID NO: 18 (GSTSGSGKPGSGEGSTKG), SEQ ID NO: 17 (GSRGGGGSGGGGSGGGGSLEMA), and SEQ ID NO: 19 (EAAAK).

[0168] Thus, in some embodiments, provided embodiments include single chain antibody fragments, e.g., scFvs, comprising one or more linkers as hereinbefore mentioned, such as a glycine / serine-rich linker, including a linker with repeats of GGGS (SEQ ID NO:20) or GGGGS (SEQ ID NO:21), such as the linkers set forth in SEQ ID NO:17, 18, 19, 22, 23, or 24. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:17. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:18. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:19. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:20. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:21. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:22. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:23. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO:24.

[0169] In some embodiments, V H The region is V L In some embodiments, the V H The region is V L In certain embodiments, the fragment, e.g., an scFv, may be carboxy-terminal to the V H A region or portion thereof, followed by a linker, followed by V L In other embodiments, the fragment, e.g., an scFv, may comprise a V L A region or portion thereof, followed by a linker, followed by V H It may include a region or part thereof.

[0170] In some embodiments, the CAR comprises a linear format. Thus, in some embodiments, the CAR comprises an anti-GPRC5D scFv and an anti-BCMA scFv. In some embodiments, the anti-GPRC5D scFv and the anti-BCMA scFv are linked by a linker. In some embodiments, the linker is represented by SEQ ID NO: 19. In some embodiments, the linker is represented by SEQ ID NO: 21. In some embodiments, the linker is represented by SEQ ID NO: 24. In some embodiments, the V of the anti-GPRC5D scFv is H and V L The regions are linked by a linker as shown in SEQ ID NO: 17. In some embodiments, the V of the anti-BCMA scFv H and V L The regions are connected by a linker shown in SEQ ID NO:17.

[0171] In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46, or has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45 or SEQ ID NO: 46. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 45, or has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the scFv provided herein comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 46, or has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 46. In some embodiments, the scFvs provided herein comprise an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:46.

[0172] The antibodies, e.g., antigen-binding fragments, in the provided CARs include human antibodies. In some embodiments of the provided human anti-GPRC5D antibodies, e.g., antigen-binding fragments, the human antibody comprises a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and / or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human heavy chain J segment. and / or contains a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment and / or contains a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. In some embodiments, the V H The portions of the regions correspond to CDR-H1, CDR-H2, and / or CDR-H3. H The portions of the regions correspond to framework region 1 (FR1), FR2, FR2, and / or FR4. L The portions of the regions correspond to CDR-L1, CDR-L2, and / or CDR-L3. L The portions of the regions correspond to FR1, FR2, FR2, and / or FR4.

[0173] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-H1 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H1 region in the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, a human antibody contains a CDR-H1 that is 100% identical to, or has no more than 1, 2, or 3 amino acid differences compared to, the corresponding CDR-H1 region in the sequence encoded by a germline nucleotide human heavy chain V segment.

[0174] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-H2 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H2 region in the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, a human antibody contains a CDR-H2 that is 100% identical to, or has no more than 1, 2, or 3 amino acid differences compared to, the corresponding CDR-H2 region in the sequence encoded by a germline nucleotide human heavy chain V segment.

[0175] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-H3 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H3 region in the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, a human antibody contains a CDR-H3 that is 100% identical to, or has no more than 1, 2, or 3 amino acid differences compared to, the corresponding CDR-H3 region in the sequence encoded by a germline nucleotide human heavy chain V segment.

[0176] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-L1 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L1 region in the sequence encoded by a germline nucleotide human light chain V segment. For example, in some embodiments, a human antibody contains a CDR-L1 that is 100% identical to, or has no more than 1, 2, or 3 amino acid differences compared to, the corresponding CDR-L1 region in the sequence encoded by a germline nucleotide human light chain V segment.

[0177] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-L2 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L2 region in the sequence encoded by a germline nucleotide human light chain V segment. For example, in some embodiments, a human antibody contains a CDR-L2 that is 100% identical to, or has no more than 1, 2, or 3 amino acid differences compared to, the corresponding CDR-L2 region in the sequence encoded by a germline nucleotide human light chain V segment.

[0178] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-L3 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L3 region in the sequence encoded by a germline nucleotide human light chain V segment. For example, in some embodiments, a human antibody contains a CDR-L3 that is 100% identical to, or has no more than 1, 2, or 3 amino acid differences compared to, the corresponding CDR-L3 region in the sequence encoded by a germline nucleotide human light chain V segment.

[0179] In some embodiments, human antibodies, e.g., antigen-binding fragments, contain framework regions that contain human germline gene segment sequences. For example, in some embodiments, human antibodies contain V and / or J segments, in which the framework regions, e.g., FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to framework regions encoded by human germline antibody segments, such as V and / or J segments. H In some embodiments, human antibodies contain framework regions, e.g., FR1, FR2, FR3, and FR4, that have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to framework regions encoded by human germline antibody segments, such as V and / or J segments. L For example, in some such embodiments, V H Area and / or V L The framework region sequences contained within the region differ by no more than 10 amino acids, for example, no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, compared to the framework region sequences encoded by human germline antibody segments.

[0180] b BCMA binding domain In some embodiments, the provided BCMA binding domain of the provided CAR contains an antibody, such as an anti-BCMA antibody, or an antigen-binding fragment thereof, that confers BCMA binding capacity to the provided CAR. In some embodiments, the CAR comprises a heavy chain variable (V) of the antibody. H ) region and / or light chain variable (V L In some embodiments, the BCMA binding domain comprises a region of the antibody, such as the (V H ) area and (V L ) region is part of a tandem dual-targeting CAR with a GPRC5D binding domain. In some embodiments, the (V H ) area and (V L) regions are connected by an interdomain linker. In some embodiments, the V H Area and V L The region comprises an scFv antibody fragment. In some embodiments, the antibody or antigen-binding domain can be or be derived from any of the anti-BCMA antibodies described (see, e.g., WO2016 / 090320 or WO2016 / 090327). Such anti-BCMA antibodies or antigen-binding fragments can be used in the provided CARs. In some embodiments, the CAR comprises a heavy chain variable (V) domain derived from an antibody described in WO2016 / 090320 or WO2016 / 090327. H ) and / or light chain variable (V L ) region.

[0181] In some embodiments, the antibody, e.g., an anti-BCMA antibody or antigen-binding fragment thereof, comprises the heavy and / or light chain variable (V H or V L ) region, or a sufficient antigen-binding portion thereof. In some embodiments, the anti-BCMA antibody, e.g., an antigen-binding fragment, comprises the V H In some embodiments, the anti-BCMA antibody, e.g., an antigen-binding fragment, contains the V domain sequence or a sufficient antigen-binding portion thereof containing the CDR-H1, CDR-H2, and / or CDR-H3 described. L In some embodiments, an anti-BCMA antibody, e.g., an antigen-binding fragment, contains a V domain sequence containing the described CDR-H1, CDR-H2, and / or CDR-H3, or a sufficient antigen-binding portion thereof containing the described CDR-L1, CDR-L2, and / or CDR-L3. H V containing the region and containing the CDR-L1, CDR-L2, and / or CDR-L3 described L Some antibodies have sequences that are at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to such sequences.

[0182] In some embodiments, the antibody or antibody fragment in the provided CAR comprises a V-like domain of any of the antibodies or antigen-binding fragments described in either WO2016 / 090320 or WO2016 / 090327. H It has an area.

[0183] In some embodiments, the CAR comprises a heavy chain variable (V) having an amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. H ) region or such a V H It comprises an antibody or antibody fragment containing CDR-H1, CDR-H2, and / or CDR-H3 appearing in the sequence.

[0184] In some embodiments, the V of the antibody or antigen-binding fragment thereof H The regions include CDR-H1, CDR-H2, and / or CDR-H3 according to Kabat numbering. H The regions include CDR-H1, CDR-H2, and / or CDR-H3 according to Chothia numbering. H The regions include CDR-H1, CDR-H2, and / or CDR-H3 according to AbM numbering.

[0185] In some embodiments, the CAR comprises a heavy chain variable (V) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 10, and a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 11. H ) area.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CDR-H1, a CDR-H2, and a CDR-H3 comprising the amino acid sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively. H Includes the area.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof comprises a V comprising the amino acid sequence set forth in SEQ ID NOs: 9, 10, and 11. H Includes the area.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the V and V sequences set forth in SEQ ID NO: 15, respectively. H The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 are contained within the region amino acid sequence.

[0189] In some embodiments of the antibodies or antigen-binding fragments thereof provided herein, V H The region includes any of the CDR-H1, CDR-H2, and CDR-H3 described above, and each of the V and V regions shown in SEQ ID NO: 15. H The region comprises framework region 1 (FR1), FR2, FR3, and / or FR4 having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to FR1, FR2, FR3, and / or FR4 contained within the amino acid sequence.

[0190] In some embodiments, the antibody or antigen-binding fragment thereof comprises the amino acid sequence set forth in SEQ ID NO: 15. H Includes the area.

[0191] In some embodiments, V H The antibody or antibody fragment in the provided CAR comprising the region further comprises a light chain or a sufficient antigen-binding portion thereof. For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises a V HArea and V L Area, or V H Area and V L In some embodiments, the V H The region sequence is V H In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.

[0192] In some embodiments, the CARs provided herein are selected from the group consisting of the V H For example, in some embodiments, the CAR comprises an antibody, such as an anti-BCMA antibody, containing any of the V and VL regions and a light chain variable region or an antigen-binding portion thereof. H Region and light chain variable (V L ) area, or V H and V L In some embodiments, the V H The region sequence is V H In some such embodiments, the antibody is an antigen-binding fragment, such as a Fab or scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.

[0193] In some embodiments, the antibody or antigen-binding fragment thereof is any of the V antibodies described in either WO2016 / 090320 or WO2016 / 090327. L It has an area.

[0194] In some embodiments, the CAR has the amino acid sequence set forth in SEQ ID NO: 16, or the V La light chain variable (V) having an amino acid sequence that has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the V region amino acid sequence; L ) region or such a V L It comprises an antibody or antibody fragment containing CDR-L1, CDR-L2, and / or CDR-L3 appearing in the sequence.

[0195] In some embodiments, the V of the antibody or antigen-binding fragment thereof L The V region comprises CDR-L1, CDR-L2, and CDR-L3 according to Kabat numbering. L The V region comprises CDR-L1, CDR-L2, and CDR-L3 according to Chothia numbering. L The regions include CDR-L1, CDR-L2, and / or CDR-L3 according to AbM numbering.

[0196] In some embodiments, the CAR comprises a light chain variable (V) comprising a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 14. L ) region.

[0197] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, and CDR-L3 comprising the amino acid sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively. L Includes the area.

[0198] In some embodiments, the antibody or antigen-binding fragment thereof comprises one of the V and V sequences set forth in SEQ ID NO: 16, respectively. L The region contains CDR-L1, CDR-L2, and CDR-L3 contained within the amino acid sequence.

[0199] The CARs provided herein include those in which the antibody, such as an anti-BCMA antibody, or antibody fragment in the provided CAR has a V sequence that has at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. H V region amino acid sequence, and an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16. L There is a CAR, which contains a region.

[0200] In some embodiments, the V of the antibody or antigen-binding fragment thereof H The regions are V and V shown in SEQ ID NO: 15, respectively. H CDR-H1, CDR-H2, and CDR-H3, each of which contains the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the amino acid sequence of the V region; and L The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 are contained within the amino acid sequence of the CDR-L1, CDR-L2, and CDR-L3 regions.

[0201] In some embodiments, the V of the antibody or antigen-binding fragment thereof H The region comprises the amino acid sequence set forth in SEQ ID NO: 15 and is a V region of the antibody or antigen-binding fragment thereof. L The V region comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the V region of the antibody or antigen-binding fragment thereof H and V L The regions comprise the amino acid sequences set forth in SEQ ID NOs: 15 and 16, respectively, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. H and V LThe present invention includes any antibody or antigen-binding fragment thereof having at least 90% sequence identity to any of the following:

[0202] For example, the V of an antibody or antigen-binding fragment thereof provided herein H and V L The regions comprise the amino acid sequences shown in SEQ ID NOs: 15 and 16, respectively.

[0203] The provided CARs include a V CAR in which the BCMA binding domain comprises the sequence set forth in SEQ ID NO: 15 or an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 15. H and comprising the sequence set forth in SEQ ID NO:16 or an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO:16. L In some embodiments, the BCMA binding domain of the provided CAR comprises a V region having CDRH1, CDRH2, and CDRH3 comprising the amino acid sequences of SEQ ID NOs: 9, 10, and 11, respectively. H and a V region having CDRL1, CDRL2, and CDRL3 comprising the amino acid sequences of SEQ ID NOs: 12, 13, and 14, respectively. L In some embodiments, the V H The region comprises the sequence shown in SEQ ID NO: 15, and V L The region comprises the sequence shown in SEQ ID NO:16.

[0204] In some embodiments, the BCMA binding domain in the provided CAR is an antibody or antigen-binding fragment thereof, which is a single-chain fragment such as a single-chain variable fragment (scFv) or diabody, or a single-domain antibody (sdAb). HIn some embodiments, the antibody or antigen-binding fragment is a single domain antibody comprising only the heavy chain variable (V H ) region and the light chain variable (V L In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable (V H ) region and the light chain variable (V L In some embodiments, a single-chain antibody fragment (e.g., an scFv) comprises a heavy chain variable (V H ) region and the light chain variable (V L The linker comprises one or more linkers connecting two antibody domains or regions, such as the nucleotide sequence (SEQ ID NO: 1) and the nucleotide sequence (SEQ ID NO: 2). The linker is typically a peptide linker, e.g., a flexible and / or soluble peptide linker. The linker may be glycine- and serine-rich, and / or, in some cases, threonine-rich. In some embodiments, the linker further comprises charged residues, such as lysine and / or glutamic acid, which may improve solubility. In some embodiments, the linker further comprises one or more prolines.

[0205] Thus, in some embodiments, the CARs provided are single chain antibody fragments, particularly human single chain antibody fragments, such as scFvs and diabodies, typically consisting of V H and V L The present invention also includes anti-BCMA antibodies, including those that include a linker connecting two antibody domains or regions, such as a β- or β-glucan domain. The linker is typically a peptide linker, e.g., a flexible and / or soluble peptide linker, such as one rich in glycine and serine.

[0206] In some embodiments, the V of the BCMA binding domain H and V L The region sequence is the V of at least one intervening GPRC5D binding domain. H and V LIn some embodiments, the extracellular antigen-binding domain of the CAR is linked by a V region sequence of the BCMA-binding domain as a loop CAR. H and V L The V region is similar to other GPRC5D binding domains. H and V L In some embodiments, the V of the BCMA binding domain has a loop structure separated by one of the V regions. H or V L At least one of the regions is connected to the V of the GPRC5D-binding domain via a linker. H and V L Directly connected to the region.

[0207] In some embodiments, the CAR comprises a loop format. In some embodiments, the V of the BCMA binding domain H or V L The V region of the GPRC5D-binding domain is separated by a linker. H or V L In some embodiments, the V of the BCMA binding domain is linked to H and V L One of the regions is the V of the BCMA binding domain. H and V L In some embodiments, the V of the GPRC5D-binding domain is linked to the other of the V H and V L One of the regions is connected to the V of the GPRC5D binding domain by a linker. H and V L In some embodiments, the linker is represented by SEQ ID NO: 17. In some embodiments, the linker is represented by SEQ ID NO: 18. In some embodiments, the linker is represented by SEQ ID NO: 19. In some embodiments, the linker is represented by SEQ ID NO: 21. In some embodiments, the linker is represented by SEQ ID NO: 22.

[0208] In some embodiments, the V of the BCMA binding domain HThe region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. L In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. L In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. L In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. H In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. H In some embodiments, the V of the BCMA binding domain is linked to H The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. H is connected to the region.

[0209] In some embodiments, the V of the BCMA binding domain L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. L In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. L In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. L In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 19. H In some embodiments, the V of the BCMA binding domain is linked toL The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 21. H In some embodiments, the V of the BCMA binding domain is linked to L The region is connected to the V of the GPRC5D binding domain by a linker shown in SEQ ID NO: 22. H is connected to the region.

[0210] In some embodiments, the extracellular antigen-binding domain of the CAR comprises the V of the BCMA binding domain. H and V L The V domains are linked directly (e.g., as scFv) by a linker, and the V domain of the GPRC5D-binding domain H and V L In some embodiments, the BCMA binding domain has a linear format in which the domains are directly linked by a linker (e.g., as an scFv). H and V L In some embodiments, the BCMA binding domain comprises, in order from N to C terminus, a V H and V L One of the regions, the linker, and V H and V L In some embodiments, the linker is that represented in SEQ ID NO: 17. Thus, in some embodiments, from N to C terminus, the BCMA binding domain comprises V H and V L One of the regions, a linker shown in SEQ ID NO: 17, and V H and V L The other of the regions.

[0211] In some embodiments, glycine and serine (and / or threonine) rich linkers comprise at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of such amino acids. In some embodiments, they comprise at least about 50%, 55%, 60%, 70%, or 75% glycine, serine, and / or threonine. In some embodiments, the linker comprises substantially entirely glycine, serine, and / or threonine. Linkers are generally about 5 to about 50 amino acids in length, typically about 10 to about 30, e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples, 10 to 25 amino acids in length. Exemplary linkers include linkers having multiple repeats of the sequence GGGGS (4GS; SEQ ID NO: 21) or GGGS (3GS; SEQ ID NO: 20), e.g., 2, 3, 4, and 5 repeats of such a sequence. Exemplary linkers include those having or consisting of the sequences set forth in SEQ ID NO: 22 (GGGGSGGGGS), SEQ ID NO: 23 (GGGGSGGGGSGGGGS), and SEQ ID NO: 24 (GGGGSGGGGSGGGGSGGGGS). Exemplary linkers further include those having or consisting of the sequences set forth in SEQ ID NO: 18 (GSTSGSGKPGSGEGSTKG), SEQ ID NO: 17 (GSRGGGGSGGGGSGGGGSLEMA), and SEQ ID NO: 19 (EAAAK).

[0212] Thus, in some embodiments, provided embodiments include single-chain antibody fragments, e.g., scFvs, comprising one or more of the linkers mentioned above, such as glycine / serine-rich linkers, including linkers with repeats of GGGS (SEQ ID NO:20), or GGGGS (SEQ ID NO:21), such as the linkers set forth in SEQ ID NOs: 17, 18, 19, 22, 23, or 24. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 18. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 20. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 21. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 22. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 23. In some embodiments, the linker comprises the sequence set forth in SEQ ID NO: 24.

[0213] In some embodiments, V H The region is V L In some embodiments, the V H The region is V L In certain embodiments, the fragment, e.g., an scFv, may be carboxy-terminal to the V H A region or portion thereof, followed by a linker, followed by V L In other embodiments, the fragment, e.g., an scFv, may comprise a V L A region or portion thereof, followed by a linker, followed by V H It may include a region or part thereof.

[0214] In some embodiments, the CAR comprises a linear format. Thus, in some embodiments, the CAR comprises an anti-GPRC5D scFv and an anti-BCMA scFv. In some embodiments, the anti-GPRC5D scFv and the anti-BCMA scFv are linked by a linker. In some embodiments, the linker is represented by SEQ ID NO: 19. In some embodiments, the linker is represented by SEQ ID NO: 21. In some embodiments, the linker is represented by SEQ ID NO: 24. In some embodiments, the V of the anti-GPRC5D scFv is H and V L The regions are linked by a linker as shown in SEQ ID NO: 17. In some embodiments, the V of the anti-BCMA scFv H and V L The regions are connected by a linker shown in SEQ ID NO:17.

[0215] In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 47 or SEQ ID NO: 48, or has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47 or SEQ ID NO: 48. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 47, or has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the scFv provided herein comprises an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 48, or has an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the scFv provided herein comprises the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the scFvs provided herein comprise an amino acid sequence having at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:48.

[0216] The antibodies, e.g., antigen-binding fragments, in the provided CARs include human antibodies. In some embodiments of the provided human anti-BCMA antibodies, e.g., antigen-binding fragments, the human antibody comprises a V segment that includes a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human heavy chain V segment, a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human heavy chain D segment, and / or a portion having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human heavy chain J segment. H and / or a V segment having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human kappa or lambda chain V segment, and / or a V segment having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence encoded by a germline nucleotide human kappa or lambda chain J segment. L In some embodiments, the V H The portions of the regions correspond to CDR-H1, CDR-H2, and / or CDR-H3. H The portions of the regions correspond to framework region 1 (FR1), FR2, FR2, and / or FR4. L The portions of the regions correspond to CDR-L1, CDR-L2, and / or CDR-L3. L The portions of the regions correspond to FR1, FR2, FR2, and / or FR4.

[0217] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-H1 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H1 region in a sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, a human antibody contains a CDR-H1 that has a sequence that is 100% identical or that has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-H1 region in a sequence encoded by a germline nucleotide human heavy chain V segment.

[0218] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-H2 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H2 region in the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, a human antibody contains a CDR-H2 that has a sequence that is 100% identical or that has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-H2 region in the sequence encoded by a germline nucleotide human heavy chain V segment.

[0219] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-H3 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H3 region in the sequence encoded by germline nucleotide human heavy chain V, D, and J segments. For example, in some embodiments, a human antibody contains a CDR-H3 that has a sequence that is 100% identical or that differs by no more than 1, 2, or 3 amino acids compared to the corresponding CDR-H3 region in the sequence encoded by germline nucleotide human heavy chain V, D, and J segments.

[0220] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-L1 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L1 region in a sequence encoded by a germline nucleotide human light chain V segment. For example, in some embodiments, a human antibody contains a CDR-L1 that has a sequence that is 100% identical or that has no more than 1, 2, or 3 amino acid differences compared to the corresponding CDR-L1 region in a sequence encoded by a germline nucleotide human light chain V segment.

[0221] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-L2 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L2 region in a sequence encoded by a germline nucleotide human light chain V segment. For example, in some embodiments, a human antibody contains a CDR-L2 that has a sequence that is 100% identical or that differs by no more than 1, 2, or 3 amino acids compared to the corresponding CDR-L2 region in a sequence encoded by a germline nucleotide human light chain V segment.

[0222] In some embodiments, a human antibody, e.g., an antigen-binding fragment, contains a CDR-L3 that has at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-L3 region in the sequence encoded by germline nucleotide human light chain V and J segments. For example, in some embodiments, a human antibody contains a CDR-L3 that has a sequence that is 100% identical or that differs by no more than 1, 2, or 3 amino acids compared to the corresponding CDR-L3 region in the sequence encoded by germline nucleotide human light chain V and J segments.

[0223] In some embodiments, human antibodies, e.g., antigen-binding fragments, contain framework regions that contain human germline gene segment sequences. For example, in some embodiments, human antibodies contain V and / or J segments, in which the framework regions, e.g., FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to framework regions encoded by human germline antibody segments, such as V and / or J segments. H In some embodiments, human antibodies contain framework regions, e.g., FR1, FR2, FR3, and FR4, that have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to framework regions encoded by human germline antibody segments, such as V and / or J segments. L For example, in some such embodiments, V H Area and / or V L The framework region sequences contained within the region differ by no more than 10 amino acids, such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids, compared to the framework region sequences encoded by human germline antibody segments.

[0224] c. Exemplary Dual-Targeting Extracellular Antigen-Binding Domains In some embodiments, the extracellular binding domain comprises a loop format. In some embodiments, from N-terminus to C-terminus, the extracellular binding domain comprises: the V of the GPRC5D binding domain H Area and V L One of the regions, V of the BCMA binding domain H Area and V L One of the regions, V of the BCMA binding domain H Area and V L the other of the regions, as well as V of the GPRC5D domain H Area and V L In some embodiments, the extracellular binding domain is the V region of the GPRC5D binding domain. H Area or VL The V region of the BCMA binding domain H Area or V L In some embodiments, the extracellular binding domain contains an interdomain linker (e.g., a first and a second interdomain linker) separating the V region from the BCMA-binding domain. In some embodiments, the first and second interdomain linkers are present and the linkers are the same. In some embodiments, the linker is any of those described herein. In some embodiments, the interdomain linker is set forth in any one of SEQ ID NOs: 19, 21, 22, or 24. In some embodiments, the extracellular binding domain is a V region of the BCMA-binding domain. H Area and V L The extracellular binding domain contains an intradomain linker separating the extracellular binding domains. In some embodiments, the intradomain linker is any of those described herein. In some embodiments, the intradomain linker is set forth in SEQ ID NO: 17 or 18. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 83, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 83. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 84, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 84. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 87, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 87. In some of any such embodiments, the extracellular antigen binding domain targets binding of the CAR for dual targeting to GPRC5D and BCMA.

[0225] In some embodiments, the extracellular binding domain comprises a loop format. In some embodiments, from N-terminus to C-terminus, the extracellular binding domain comprises: the V of the BCMA binding domain; H Area and V L One of the regions, V of the GPRC5D binding domain H Area and V L One of the regions, V of the GPRC5D binding domain H Area and V L the other of the regions, as well as the V of the BCMA binding domain H Area and V L In some embodiments, the extracellular binding domain is the V region of the BCMA binding domain. H Area or V L The V region of the GPRC5D binding domain H Area or V L In some embodiments, the extracellular binding domain comprises an interdomain linker (e.g., first and second interdomain linkers) separating the V region from the GPRC5D-binding domain. In some embodiments, the linker is any of those described herein. In some embodiments, a first and second interdomain linker are present, and the linkers are the same. In some embodiments, the interdomain linker is set forth in any one of SEQ ID NOs: 19, 21, 22, or 24. In some embodiments, the extracellular binding domain is a V region of the GPRC5D-binding domain. H Area and V LThe extracellular binding domain contains an intradomain linker separating the extracellular binding domains. In some embodiments, the intradomain linker is any of those described herein. In some embodiments, the intradomain linker is set forth in SEQ ID NO: 17 or 18. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 81, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 81. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 82, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 82. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 85, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 85. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 86, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 86. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 88, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 88. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 89, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 89.In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 90, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 90. In some of any such embodiments, the extracellular antigen binding domain targets binding of the CAR for dual targeting to GPRC5D and BCMA.

[0226] In some embodiments, the extracellular binding domain comprises a linear format. In some embodiments, from N-terminus to C-terminus, the extracellular binding domain comprises: the V of the GPRC5D binding domain H Area and V L One of the regions, V of the GPRC5D binding domain H Area and V L The other region, V of the BCMA binding domain H Area and V L one of the regions, as well as the V of the BCMA binding domain H Area and V L In some embodiments, the extracellular binding domain is the V region of the GPRC5D binding domain. H Area and V L In some embodiments, the extracellular binding domain comprises an intradomain linker separating the V region of the BCMA binding domain. H Area and V L In some embodiments, the extracellular binding domain comprises an intradomain linker separating the V region of the GPRC5D-binding domain. In some embodiments, the intradomain linker is any of those described herein. In some embodiments, the intradomain linker is set forth in SEQ ID NO: 17 or 18. In some embodiments, the extracellular binding domain comprises an intradomain linker separating the V region of the GPRC5D-binding domain. H Area or V L The V region of the BCMA binding domain H Area or V LThe extracellular binding domain contains an inter-domain linker separating the domains. In some embodiments, the linker is any of those described herein. In some embodiments, a first and a second inter-domain linker are present, and the linkers are the same. In some embodiments, the inter-domain linker is set forth in any one of SEQ ID NOs: 19, 21, 22, or 24. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 77, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 77. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 78, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 78. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 79, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 79. In some embodiments, the extracellular binding domain has a sequence of amino acids set forth in SEQ ID NO: 80, or a sequence of amino acids exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 80. In some of any such embodiments, the extracellular antigen binding domain targets binding of the CAR for dual targeting to GPRC5D and BCMA.

[0227] In some embodiments, the extracellular binding domain comprises a linear format. In some embodiments, from N-terminus to C-terminus, the extracellular binding domain comprises: the V of the BCMA binding domain; H Area and V L One of the regions, V of the BCMA binding domainH Area and V L The other region, V of the GPRC5D binding domain H Area and V L one of the regions, as well as V of the GPRC5D-binding domain H Area and V L In some of any such embodiments, the extracellular antigen-binding domain targets binding of the CAR for dual targeting to GPRC5D and BCMA.

[0228] In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 77-90. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:90.

[0229] In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 77-80, 83, 84, and 87. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 7 ...8. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 87.

[0230] In some embodiments, the extracellular binding domain is designed such that the GPRC5D targeting binding domain is proximal to the transmembrane domain.

[0231] In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 83, 84, and 87. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 84. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 87.

[0232] In some embodiments, the extracellular binding domain is designed such that the BCMA targeting binding domain is proximal to the transmembrane domain.

[0233] In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 77 to 80. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 80.

[0234] In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 81, 82, 85, 86, 88, 89, and 90. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO: 90.

[0235] 2. Spacer In some embodiments, a recombinant receptor such as a CAR comprising an extracellular antigen-binding domain provided herein further comprises a spacer. In some embodiments, the spacer is or comprises at least a portion of an immunoglobulin constant region or a variant or modified version thereof. In some embodiments, the portion of the immunoglobulin constant region comprises a hinge region, for example, an IgG4 hinge region, and / or a C H 1. C H 2. C HIn some embodiments, the constant region or portion is a human IgG, such as IgG4 or IgG1. In some embodiments, the portion of the constant region comprises an antigen-binding domain or portion thereof (e.g., the V of the GPRC5D-binding domain or the BCMA-binding domain). H or V L The spacer serves as a spacer region between the CAR-expressing cell (e.g., a CAR-expressing T cell) and the transmembrane domain. In some embodiments, the length of the spacer is adjusted to optimize the biophysical synaptic distance between the CAR-expressing cell, such as a CAR-expressing T cell, and the target of the CAR, such as a GPRC5D- or BCMA-expressing cell. In some embodiments, the CAR is expressed by a T cell, and the length of the spacer is adjusted to a length that is compatible with T cell activation or that optimizes CAR T cell performance.

[0236] In some embodiments, the spacer can be of a length that results in increased cellular responsiveness after antigen binding compared to no spacer or compared to an alternative spacer of a different length (e.g., a shorter length). In some examples, the spacer is about 12 amino acids in length, or 12 amino acids or less in length. In some embodiments, the spacer is at least 100 amino acids in length, e.g., at least 110, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids in length. Exemplary spacers include those having at least about 10 to 300 amino acids, about 10 to 200 amino acids, about 50 to 175 amino acids, about 50 to 150 amino acids, about 10 to 125 amino acids, about 50 to 100 amino acids, about 100 to 300 amino acids, about 100 to 250 amino acids, about 125 to 250 amino acids, or about 200 to 250 amino acids, including any integer between either end of the recited range. In some embodiments, the spacer region is at least about 12 amino acids in length, at least about 119 amino acids, at least about 125 amino acids, at least about 200 amino acids, or at least about 220 amino acids, or at least about 225 amino acids.

[0237] In some embodiments, the spacer is 125 to 300 amino acids in length, 125 to 250 amino acids in length, 125 to 230 amino acids in length, 125 to 200 amino acids in length, 125 to 180 amino acids in length, 125 to 150 amino acids in length, 150 to 300 amino acids in length, 150 to 250 amino acids in length, 150 to 230 amino acids in length, 150 to 200 amino acids in length, 150 to 180 amino acids in length, 180 to 300 amino acids in length, 180 to 250 amino acids in length, 180 to 230 amino acids in length, 180 to 200 amino acids in length, 200 to 300 amino acids in length, 200 to 250 amino acids in length, 200 to 230 amino acids in length, 230 to 300 amino acids in length, 230 to 250 amino acids in length, or 250 to 300 amino acids in length. In some embodiments, the spacer is at least or at least about, or about, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, or 229 amino acids in length, or any of the lengths in between.

[0238] Exemplary spacers are IgG hinge alone, C H 2 and C H IgG hinge linked to one or more of the three domains, or C H In some embodiments, the IgG hinge comprises an IgG hinge linked to three domains. H 2, and / or C H 3 can be derived from all or part of IgG4 or IgG2, such as all or part of human IgG4 or human IgG2. In some embodiments, the spacer comprises a hinge, C ... H 2, and / or C HIn some embodiments, the hinge region comprises all or part of an IgG4 hinge region and / or an IgG2 hinge region, where the IgG4 hinge region is optionally a human IgG4 hinge region and the IgG2 hinge region is optionally a human IgG2 hinge region; H 2 regions are IgG4 C H 2 domain and / or IgG2 C H IgG4 C H 2 regions are optionally human IgG4 C H 2-domain and IgG2 C H 2 regions are optionally human IgG2 C H 2 regions; and / or C H 3 regions are IgG4 C H 3 domain and / or IgG2 C H IgG4 C3 region, in whole or in part, H 3 regions are optionally human IgG4 C H 3-domain and IgG2 C H 3 regions are optionally human IgG2 C H In some embodiments, the hinge, C H 2, and C H 3 is the hinge region derived from IgG4, C H 2, and C H In some embodiments, the hinge region is chimeric and comprises hinge regions derived from human IgG4 and human IgG2; and C H The two regions are chimeric and are derived from human IgG4 and human IgG2. H 2 regions; and / or C H The three regions are chimeric and are derived from human IgG4 and human IgG2. H In some embodiments, the spacer comprises three regions: an IgG4 / 2 chimeric hinge or a modified IgG4 hinge comprising at least one amino acid substitution compared to the human IgG4 hinge region; H 2 domains; and human IgG4 C H Includes three areas.

[0239] In some embodiments, the spacer may be derived from all or part of IgG4 and / or IgG2 and may contain mutations, such as one or more single amino acid mutations, in one or more domains. In some examples, the amino acid modification is a proline (P) to serine (S) substitution in the hinge region of IgG4. In some embodiments, the amino acid modification is a C nucleotide sequence of the full-length IgG4 Fc sequence shown in SEQ ID NO: 75. H N177Q mutation at position 177 in the IgG2 Fc region, or C of the full-length IgG2 Fc shown in SEQ ID NO: 76 H In some embodiments, the spacer is a glutamine (Q) to asparagine (N) substitution to reduce glycosylation heterogeneity, such as N176Q at position 176 in the 2 region. In some embodiments, the spacer is an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric hinge; H 2 region; and IgG4 C H In some embodiments, the spacer is or comprises three regions. In some embodiments, the spacer is about 228 amino acids in length. In some embodiments, the spacer is represented by SEQ ID NO: 27. In some embodiments, the spacer comprises the following amino acid sequence: ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 27)

[0240] In some embodiments, the spacer is encoded by a polynucleotide that has been optimized for codon expression and / or to remove splice sites, such as cryptic splice sites. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:49. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:50. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:73. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:74.

[0241] Additional exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol. Res., 3(2):125-135, or International Patent Application Publication No. WO2014031687. In some embodiments, the nucleic acid sequence of the spacer is optimized to reduce RNA heterogeneity during expression. In some embodiments, the nucleic acid sequence of the spacer is optimized to reduce cryptic splice sites or to reduce the likelihood of splicing events at splice sites.

[0242] In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 25 and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 51. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 52. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 54 and is encoded by the polynucleotide sequence set forth in SEQ ID NO: 53.

[0243] In some embodiments, the spacer has an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 27. In some embodiments, the spacer has the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, the spacer is encoded by a polynucleotide sequence set forth in SEQ ID NO: 49, 50, 73, or 74, or a polynucleotide that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to SEQ ID NO: 49, 50, 73, or 74.

[0244] In some embodiments, the spacer is encoded by a polynucleotide, optionally optimized for codon usage and / or to reduce RNA heterogeneity. Methods for reducing RNA heterogeneity, such as by removing cryptic splice donor and / or acceptor sites, are described below. Observations have shown that cryptic splice donor / acceptor sites exist in the spacer region of certain immunoglobulin spacers when present in a CAR. In some embodiments, the spacer in the provided CAR is encoded by a polynucleotide in which one or more cryptic splice donor and / or acceptor sites have been removed and / or modified to reduce heterogeneity after the RNA transcribed from the construct, such as an mRNA, is expressed in a cell. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO:49. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO:50. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO:73. In some embodiments, the spacer is encoded by the nucleotide sequence set forth in SEQ ID NO:74.

[0245] 3. Transmembrane domains and intracellular signaling components The extracellular antigen-binding domains (i.e., GPRC5D and BCMA-binding domains) are generally linked to one or more signaling components, such as, in the case of CARs, signaling components that mimic activation via an antigen receptor complex, such as a TCR complex, and / or signal through another cell surface receptor. Thus, in some embodiments, the GPRC5D-binding domain or component thereof, or the BCMA-binding domain or component thereof (e.g., an antibody or antigen-binding fragment thereof) is linked to an intracellular signaling domain that includes one or more transmembrane domains, such as those described herein, and one or more intracellular components, such as those described herein. In some embodiments, the V of the binding domain closest to the cell membrane is linked to one or more signaling components, such as, for example, a BCMA-binding domain or component thereof (e.g., an antibody or antigen-binding fragment thereof). H or V L is linked to the transmembrane domain. Typically, a binding domain or a component thereof (e.g., V H Area or V L The extracellular domain (e.g., a transmembrane domain sequence) is indirectly linked to the transmembrane domain via a spacer sequence (e.g., Section I.2). In some embodiments, the transmembrane domain is fused to the extracellular domain. In certain embodiments, a transmembrane domain that is naturally associated with one of the domains of a receptor, e.g., a CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to transmembrane domains of the same or different membrane surface proteins and to minimize interactions with other members of the receptor complex.

[0246] In some embodiments, the transmembrane domain is derived from either a natural or synthetic source. If the source is natural, in some embodiments, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane domains include those derived from the alpha, beta, or zeta chain of the T cell receptor, CD3 epsilon, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and / or CD154 (i.e., at least these transmembrane domains). For example, in some embodiments, the transmembrane domain can be a CD28 transmembrane domain comprising the sequence of amino acids set forth in SEQ ID NO: 18, such as may be encoded by the nucleic acid sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56. Alternatively, in some embodiments, the transmembrane domain can be synthetic. In some embodiments, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine may be present at each end of the synthetic transmembrane domain. In some embodiments, the linkage is by a linker, spacer, and / or transmembrane domain.

[0247] The intracellular signaling domain can mimic or approximate signaling through a natural antigen receptor, signaling through such a receptor in combination with a costimulatory receptor, and / or signaling through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, e.g., a linker 2 to 10 amino acids in length, such as one containing glycine and serine, e.g., a glycine-serine doublet, is present to form the link between the transmembrane domain of the CAR and the intracellular signaling domain.

[0248] Receptors, e.g., CARs, generally comprise an intracellular signaling region, including at least one intracellular signaling component or components. In some embodiments, the receptor comprises an intracellular component or signaling domain of the TCR complex, such as a TCR CD3 chain, e.g., the CD3 zeta (CD3-ζ) chain, which mediates T cell activation and cytotoxicity. Thus, in some embodiments, the GPRC5D or BCMA-binding antibody is linked to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further comprises a portion of one or more additional molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some embodiments, the CAR comprises a chimeric molecule between CD3 zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0249] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling domain of the CAR stimulates and / or activates at least one of the normal effector functions or responses of an immune cell, e.g., a T cell engineered to express the CAR. For example, in some contexts, the CAR induces a T cell function, such as cytolytic activity or T helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, e.g., if it transmits an effector function signal. In some embodiments, the intracellular signaling domain or domains comprise the cytoplasmic sequence of a T cell receptor (TCR), and in some embodiments also include the sequence of a co-receptor that naturally cooperates with such receptor to initiate signal transduction following antigen receptor engagement and / or a derivative or variant of such molecule, and / or any synthetic sequence having the same functionality.

[0250] In the context of natural TCR, full activation generally requires not only TCR-mediated signaling but also costimulatory signals. Thus, in some embodiments, the CAR also includes components for generating secondary or costimulatory signals to promote full activation. In other embodiments, the CAR does not include components for generating costimulatory signals. In some embodiments, an additional CAR is expressed in the same cell and provides components for generating secondary or costimulatory signals.

[0251] T cell activation is described in some embodiments as mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences). In some embodiments, a CAR comprises one or both of these classes of cytoplasmic signaling sequences.

[0252] In some embodiments, the CAR comprises a primary cytoplasmic signaling sequence that regulates primary stimulation and / or activation of the TCR complex. The stimulatory primary cytoplasmic signaling sequence may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, CD3 gamma, CD3 delta, and CD3 epsilon. In some embodiments, the intracellular signaling region of the CAR contains a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta. In some embodiments, the CD3 zeta comprises the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the CD3 zeta is encoded by the nucleic acid sequence set forth in SEQ ID NO: 55 or SEQ ID NO: 56.

[0253] In some embodiments, the CAR comprises a signaling domain (e.g., an intracellular or cytoplasmic signaling domain) and / or a transmembrane portion of a costimulatory domain, such as a T cell costimulatory domain. Exemplary costimulatory molecules include CD28, 4-1BB, OX40, DAP10, and ICOS. For example, the costimulatory molecule may be derived from 4-1BB and may comprise the amino acid sequence set forth in SEQ ID NO:29. In some embodiments, 4-1BB is encoded by the nucleotide sequence set forth in SEQ ID NO:57 or SEQ ID NO:58. In some cases, the costimulatory molecule may be derived from CD28 and may comprise the amino acid sequence set forth in SEQ ID NO:100. In some embodiments, the same CAR comprises both a stimulatory or activating component (e.g., a cytoplasmic signaling sequence) and a costimulatory component.

[0254] In some embodiments, the stimulatory or activating component is contained within one CAR, while the costimulatory component is provided by another CAR that recognizes a different antigen. In some embodiments, the CAR comprises an activating or stimulatory CAR and a costimulatory CAR, both of which are expressed on the same cell (see WO2014 / 055668). In some embodiments, the GPRC5D-targeting CAR is a stimulatory or activating CAR; in other embodiments, it is a costimulatory CAR. In some embodiments, the cell further comprises an inhibitory CAR (e.g., an iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013)), such as a CAR that recognizes an antigen other than GPRC5D, thereby reducing or inhibiting the stimulatory or activating signal transmitted through the GPRC5D-targeting CAR upon binding of the inhibitory CAR to its ligand), e.g., to reduce off-target effects.

[0255] In certain embodiments, the extracellular signaling region comprises the transmembrane and signaling domains of CD28 linked to the CD3 (e.g., CD3 zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and 4-1BB (CD137; TNFRSF9) costimulatory domain linked to the CD3 zeta intracellular domain.

[0256] In some embodiments, the CAR includes one or more, e.g., two or more, costimulatory domains and stimulatory or activation domains, e.g., primary activation domains, in the cytoplasmic portion. Exemplary CARs include the intracellular components of CD3 zeta, CD28, and 4-1BB.

[0257] In some embodiments, provided embodiments of anti-GPRC5D CARs contain an extracellular antigen-binding domain containing any of the anti-GPRC5D antibodies or antigen-binding fragments described herein, such as in Section I.1; a spacer comprising an IgG4 / 2 chimeric hinge or modified IgG4 hinge, an IgG2 / 4 chimeric CH2 region, and an IgG4 CH3 region, such as one approximately 228 amino acids in length, or a spacer as set forth in SEQ ID NO: 27, such as one encoded by the nucleic acid sequence set forth in any of SEQ ID NOs: 49, 50, 73, and 74; a transmembrane domain, such as a transmembrane domain derived from human CD28; and an intracellular signaling region comprising the cytoplasmic signaling domain of the CD3 zeta (CD3ζ) chain and the intracellular signaling domain of a T cell costimulatory molecule. Also provided are polynucleotides encoding such chimeric antigen receptors. In some embodiments, the transmembrane domain is or comprises the sequence set forth in SEQ ID NO: 28. In some embodiments, the intracellular signaling domain of the T cell costimulatory molecule is the intracellular signaling domain of human CD28, human 4-1BB, or human ICOS, or a signaling portion thereof. In some embodiments, the intracellular signaling domain is the intracellular signaling domain of human 4-1BB. In some embodiments, the intracellular signaling domain is or comprises the sequence set forth in SEQ ID NO:29. In some embodiments, the cytoplasmic signaling domain is a CD3 zeta cytoplasmic signaling domain, such as that set forth in SEQ ID NO:30. In some embodiments, the intracellular signaling region comprises the sequence set forth in SEQ ID NO:30 and SEQ ID NO:29.

[0258] 4. Exemplary CAR In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: V of the GPRC5D-binding domain L region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 19, a V of the BCMA binding domain Hregion, a linker shown in SEQ ID NO: 17, and the V of the BCMA binding domain L region.

[0259] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: V of the GPRC5D-binding domain L region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 24, a V of the BCMA binding domain H region, a linker shown in SEQ ID NO: 17, and the V of the BCMA binding domain L region.

[0260] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: V of the GPRC5D-binding domain H region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 21, a V of the BCMA binding domain H region, a linker shown in SEQ ID NO: 17, and the V of the BCMA binding domain L region.

[0261] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: V of the GPRC5D-binding domain H region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 24, a V of the BCMA binding domain L region, a linker shown in SEQ ID NO: 17, and the V of the BCMA binding domain H region.

[0262] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: a V of a BCMA-binding domain; H region, a linker shown in SEQ ID NO: 19, V of the GPRC5D binding domain Lregion, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 19, and the V of the BCMA binding domain L region.

[0263] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: a V of a BCMA-binding domain; L region, a linker shown in SEQ ID NO: 19, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 24, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 19, and the V of the BCMA binding domain H region.

[0264] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: V of the GPRC5D-binding domain H region, a linker shown in SEQ ID NO: 21, a V of the BCMA binding domain L region, a linker shown in SEQ ID NO: 17, a V of the BCMA binding domain H region, a linker shown in SEQ ID NO: 21, and the V of the GPRC5D binding domain L region.

[0265] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: V of the GPRC5D-binding domain L region, a linker shown in SEQ ID NO: 21, a V of the BCMA binding domain H region, a linker shown in SEQ ID NO: 17, a V of the BCMA binding domain L region, a linker shown in SEQ ID NO: 21, and the V of the GPRC5D binding domain H region.

[0266] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: a V of a BCMA-binding domain; Lregion, a linker shown in SEQ ID NO: 21, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 21, and the V of the BCMA binding domain H region.

[0267] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: a V of a BCMA-binding domain; L region, a linker shown in SEQ ID NO: 21, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 21, and the V of the BCMA binding domain H region.

[0268] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: V of the GPRC5D-binding domain H region, a linker shown in SEQ ID NO: 22, a V of the BCMA binding domain L region, a linker shown in SEQ ID NO: 17, a V of the BCMA binding domain H region, a linker shown in SEQ ID NO: 22, and the V of the GPRC5D-binding domain L region.

[0269] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: a V of a BCMA-binding domain; H region, a linker shown in SEQ ID NO: 22, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 22, and the V of the BCMA binding domain L region.

[0270] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: a V of a BCMA-binding domain; L region, a linker shown in SEQ ID NO: 22, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 17, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 22, and the V of the BCMA binding domain H region.

[0271] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising, in N- to C-terminus, the following: a V of a BCMA-binding domain; L region, a linker shown in SEQ ID NO: 22, V of the GPRC5D binding domain H region, a linker shown in SEQ ID NO: 24, V of the GPRC5D binding domain L region, a linker shown in SEQ ID NO: 22, and the V of the BCMA binding domain H region.

[0272] In some embodiments, the CAR comprises an extracellular antigen-binding domain comprising a spacer set forth in SEQ ID NO: 27. In some embodiments, the CAR comprises a transmembrane domain set forth in SEQ ID NO: 28. In some embodiments, the CAR comprises an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 29 or 30.

[0273] In some embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 31-44, or is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 105-120. In some embodiments, the CAR comprises the amino acid sequence set forth in any one of SEQ ID NOs: 31-44. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in any one of SEQ ID NOs: 105-120.

[0274] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 105. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 31 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 105. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 105. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 31. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0275] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 106. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 32 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 106. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 106. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0276] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 107. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 33 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 107. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 107. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0277] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 108. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 34 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 108. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 108. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0278] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 109. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 35 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 109. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 109. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0279] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 110. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 36 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 110. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 36. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 110. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0280] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 111. In some embodiments, the CAR is encoded by a nucleotide sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 119. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 37 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 111. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 37 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 119. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 37. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 111. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 119. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0281] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 112. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 38 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 112. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 38. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 112. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0282] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 113. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 39. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 113. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 112. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0283] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 40. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 114. In some embodiments, the CAR is encoded by a nucleotide sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 120. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 40 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 114. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 40 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 120. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 114. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 120. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0284] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 115. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 115. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 120. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0285] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 116. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 42 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 116. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 116. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0286] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 117. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 43 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 117. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 117. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0287] In some embodiments, the CAR comprises a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44. In some embodiments, the CAR is encoded by a nucleotide sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 118. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 44 or is encoded by the nucleotide sequence set forth in SEQ ID NO: 118. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 44. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 118. In some of any such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA, such as those expressed on the surface of cells (e.g., cancer cells, such as plasma cells with multiple myeloma).

[0288] 5. Exemplary Features In some or any of the provided embodiments, the bispecific CAR and / or GPRC5D binding domain, antibody, or antigen-binding fragment specifically binds to GPRC5D, such as GPRC5D on the surface of multiple myeloma plasma cells. In some embodiments, the binding can be to human GPRC5D, mouse GPRC5D protein, or non-human primate (e.g., cynomolgus monkey) GPRC5D protein. In some embodiments, the provided bispecific CAR and / or GPRC5D binding domain binds to human GPRC5D protein. The observation that an antibody or other binding molecule binds to GPRC5D protein or specifically binds to GPRC5D protein does not necessarily mean that it binds to GPRC5D protein of all species. For example, in some embodiments, binding characteristics to GPRC5D protein, such as the ability to specifically bind to it and / or the ability to compete with a reference antibody in binding to it, and / or the ability to bind with a particular affinity or compete to a particular degree, refer in some embodiments to the ability with respect to human GPRC5D protein, and the antibody may not have this characteristic with respect to GPRC5D protein of another species, such as mouse.

[0289] In some embodiments, the antibody specifically binds to human GPRC5D protein, such as at an epitope or region of the human GPRC5D protein, e.g., a human GPRC5D protein comprising the amino acid sequence of SEQ ID NO: 59 (Uniprot Q9NZD1), or an allelic variant or splice variant thereof.

[0290] In some embodiments, the degree of binding of an anti-GPRC5D antibody or antigen-binding domain or CAR to an unrelated non-GPRC5D protein, such as a non-human GPRC5D protein or another non-GPRC5D protein, is less than about 10% of the binding of the antibody or antigen-binding domain or CAR to human GPRC5D protein or human membrane-bound GPRC5D, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody or antigen-binding domain in the provided CAR is an antibody or antigen-binding domain or CAR whose binding to mouse GPRC5D protein is less than about 10% of the binding of the antibody to human GPRC5D protein. In some embodiments, the antibody or antigen-binding domain in the provided CAR is an antibody whose binding to cynomolgus monkey GPRC5D protein is less than about 10% of the binding of the antibody to human GPRC5D protein. In some embodiments, the antibody or antigen-binding domain in the provided CAR includes an antibody whose binding to cynomolgus monkey GPRC5D protein and / or mouse GPRC5D protein is similar or nearly identical to the antibody's binding to human GPRC5D protein.

[0291] In some embodiments, the antibodies in the provided CARs can bind to a GPRC5D protein, such as a human GPRC5D protein, with at least a particular affinity as measured by any of a number of known methods. In some embodiments, affinity is measured using the equilibrium dissociation constant (K D ) and in some embodiments, affinity is represented by EC 50 It is expressed as:

[0292] Various assays are known for assessing binding affinity and / or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand (e.g., an antigen such as GPRC5D protein). It is within the level of one skilled in the art to determine the binding affinity of a binding molecule, e.g., an antibody, to an antigen, e.g., GPRC5D, such as human GPRC5D, cynomolgus monkey GPRC5D, or mouse GPRC5D, for example, using any of a number of binding assays well known in the art. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex of two proteins (e.g., an antibody or fragment thereof, and an antigen such as GPRC5D protein) using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0293] In some or any of the provided embodiments, the bispecific CAR and / or BCMA binding domain, antibody, or antigen-binding fragment specifically binds to BCMA, such as BCMA on the surface of multiple myeloma plasma cells. In some embodiments, binding can be to human BCMA, mouse BCMA protein, or non-human primate (e.g., cynomolgus monkey) BCMA protein. In some embodiments, the provided bispecific CAR and / or BCMA binding domain binds to human BCMA protein. The observation that an antibody or other binding molecule binds to or specifically binds to BCMA protein does not necessarily mean that it binds to BCMA protein of all species. For example, in some embodiments, binding characteristics to BCMA protein, such as the ability to specifically bind thereto and / or compete with a reference antibody in binding thereto, and / or the ability to bind with a particular affinity or compete to a particular degree, refer to the ability in some embodiments with respect to human BCMA protein; the antibody may not have this characteristic with respect to BCMA protein of another species, such as mouse.

[0294] In some embodiments, the antibody specifically binds to a human BCMA protein, e.g., an epitope or region of a human protein, e.g., a human BCMA protein comprising the amino acid sequence of SEQ ID NO: 60 (Uniprot Q02223), or an allelic or splice variant thereof.

[0295] In some embodiments, the extent of binding of an anti-BCMA antibody or antigen-binding domain or CAR to an unrelated non-BCMA protein, such as a non-human BCMA protein or other non-BCMA protein, is less than about 10% of the binding of the antibody or antigen-binding domain or CAR to human BCMA protein or human membrane-bound BCMA, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody or antigen-binding domain in a provided CAR is an antibody or antigen-binding domain or CAR that binds to mouse BCMA protein at less than about 10% of the binding of the antibody to human BCMA protein. In some embodiments, the antibody or antigen-binding domain in a provided CAR is an antibody or antigen-binding domain or CAR that binds to cynomolgus BCMA protein at less than about 10% of the binding of the antibody to human BCMA protein. In some embodiments, the antibody or antigen-binding domain in a provided CAR is an antibody that binds to cynomolgus BCMA protein and / or mouse BCMA protein at similar or nearly identical to the binding of the antibody to human BCMA protein.

[0296] In some embodiments, the antibodies in the provided CARs can bind to a BCMA protein, such as a human BCMA protein, with at least a particular affinity as measured by any of a number of known methods. In some embodiments, affinity is measured using the equilibrium dissociation constant (K D ) and in some embodiments, affinity is represented by EC 50 It is expressed as:

[0297] A variety of assays are known for assessing binding affinity and / or determining whether a binding molecule (e.g., an antibody or fragment thereof) specifically binds to a particular ligand (e.g., an antigen such as a BCMA protein). It is within the level of one of ordinary skill in the art to determine the binding affinity of a binding molecule, e.g., an antibody, to an antigen, e.g., BCMA, such as human BCMA or cynomolgus BCMA or mouse BCMA, such as using any of a number of binding assays well known in the art. For example, in some embodiments, a BIAcore® instrument can be used to determine the binding kinetics and constants of a complex of two proteins (e.g., an antibody or fragment thereof, and an antigen such as a BCMA protein) using surface plasmon resonance (SPR) analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0298] SPR measures changes in the concentration of molecules at a sensor surface as they bind to or dissociate from the surface. The change in SPR signal is directly proportional to the change in mass concentration near the surface, allowing for measurement of the binding kinetics between two molecules. The dissociation constant of the complex can be determined by monitoring the change in refractive index over time as a buffer passes through the chip. Other suitable assays for measuring the binding of one protein to another include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs), or determining binding by monitoring changes in the spectroscopic or optical properties of the proteins via fluorescence, UV absorbance, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include, but are not limited to, Western blots, ELISAs, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for detecting binding of expressed polynucleotides or proteins.

[0299] In some embodiments, the binding molecule, e.g., an antibody or fragment thereof, or an antigen-binding domain of a CAR, binds to an antigen, e.g., a GPRC5D protein or an epitope thereof, in an amount of 10 5 M -1 Affinity or K equal to or greater than A (i.e., the equilibrium binding constant of a particular binding interaction in units of 1 / M, which is the on-rate [k on or k a ] off rate [k off or k d In some embodiments, the antigen-binding domain of the antibody or fragment thereof or CAR binds, e.g., specifically binds, at a ratio equal to 10 -5 K equal to or less than M D (i.e., the equilibrium dissociation constant of a particular binding interaction, in units of M, and the off-rate [k off or k d ] on speed [kon or k a ] (assuming a bimolecular interaction) exhibits binding affinity for the peptide epitope. For example, the equilibrium dissociation constant K D is 10 -7 M to 10 -11 M, 10 -8 M to 10 -10 M, or 10 -9 M to 10 -10 M, etc., 10 -5 M to 10 -13 The on-rate (association rate constant; k on or k a ; units 1 / M) and off-rate (dissociation rate constant; k off or k d ; units 1 / s) can be determined using any assay method known in the art, for example, surface plasmon resonance (SPR).

[0300] In some embodiments, the binding affinity (EC 50) and / or the dissociation constant is from about 0.01 nM to about 500 nM, from about 0.01 nM to about 400 nM, from about 0.01 nM to about 100 nM, from about 0.01 nM to about 50 nM, from about 0.01 nM to about 10 nM, from about 0.01 nM to about 1 nM, from about 0.01 nM to about 0.1 nM, from about 0.1 nM to about 500 nM, from about 0.1 nM to about 400 nM, from about 0.1 nM to about 100 nM, from about 0.1 nM to about 50 nM, to about 10 nM, about 0.1 nM to about 1 nM, about 0.5 nM to about 200 nM, about 1 nM to about 500 nM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 1 nM to about 10 nM, about 2 nM to about 50 nM, about 10 nM to about 500 nM, about 10 nM to about 100 nM, about 10 nM to about 50 nM, about 50 nM to about 500 nM, about 50 nM to about 100 nM, or about 100 nM to about 500 nM. In certain embodiments, the binding affinity (EC50) and / or equilibrium dissociation constant KD of an antibody to a GPRC5D protein, such as a human GPRC5D protein, is at, or less than, or about: 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibody binds to a GPRC5D protein, such as a human GPRC5D protein, with a binding affinity of the nanomolar or less, for example, a binding affinity of less than about 1 nM, such as about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, or about 0.1 nM or less.

[0301] In some embodiments, binding affinity may be classified as high or low affinity. In some cases, a binding molecule (e.g., an antibody or fragment thereof) or an antigen-binding domain of a CAR exhibiting low to moderate binding affinity may be 10 7 M -1 Up to 10 6 M -1Up to 10 5 M -1 K up to A In some cases, a binding molecule (e.g., an antibody or fragment thereof) that exhibits high binding affinity for a particular epitope exhibits a binding affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 K A In some embodiments, the binding affinity (EC ) of a binding molecule, e.g., an anti-GPRC5D antibody or fragment thereof, or an antigen-binding domain of a CAR, to a GPRC5D protein is 50 )) and / or the equilibrium dissociation constant K D is about 0.01 nM to about 1 μM, 0.1 nM to 1 μM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM. In certain embodiments, the binding affinity (EC 50 ) and / or the equilibrium dissociation constant K Dis at, about, or less than about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. The degree of affinity of a particular antibody can be compared to the affinity of a known antibody, such as a reference antibody.

[0302] In some embodiments, the binding affinity of a binding molecule, such as an anti-GPRC5D antibody or an antigen-binding domain of a CAR, to different antigens, for example, GPRC5D proteins from different species, can be compared to determine species cross-reactivity. For example, species cross-reactivity can be classified as high cross-reactivity or low cross-reactivity. In some embodiments, the equilibrium dissociation constant K for different antigens, for example, GPRC5D proteins from different species, such as humans, cynomolgus monkeys, or mice, can be compared. D can be compared to determine species cross-reactivity. In some embodiments, the species cross-reactivity of an anti-GPRC5D antibody or antigen-binding domain of a CAR can be high, e.g., the anti-GPRC5D antibody binds to human GPRC5D and species variant GPRC5D to a similar extent, e.g., the K D and K against species variant GPRC5D D The ratio of K to species variant GPRC5D is about 1. In some embodiments, the species cross-reactivity of the anti-GPRC5D antibody or the antigen-binding domain of the CAR may be low, for example, the anti-GPRC5D antibody has high affinity for human GPRC5D but low affinity for species variant GPRC5D, or vice versa. For example, the K D and K against human GPRC5D D is 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, or more, and the anti-GPRC5D antibody has low species cross-reactivity. The degree of species cross-reactivity can be compared to the species cross-reactivity of a known antibody, such as a reference antibody.

[0303] Some provided bispecific CARs exhibit antigen-dependent activity or signal transduction, i.e., signal transduction activity that is not measurable or at background level in the absence of antigen, such as GPRC5D.Thus, in some embodiments, provided CARs do not exhibit sustained signal transduction or antigen-dependent activity or signal transduction in the absence of antigen, such as GPRC5D, or only exhibit below background or tolerable or low levels.In some embodiments, the cells that express provided bispecific CARs exhibit biological activity or function, including cytotoxic activity, cytokine production, and proliferation ability.

[0304] In some embodiments, the binding molecule, e.g., an antibody or fragment thereof, or an antigen-binding domain of a CAR, binds to an antigen, e.g., a BCMA protein (e.g., SEQ ID NO: 60) or an epitope thereof, in an amount of 10 5 M -1 Affinity or K equal to or greater than A (i.e., the equilibrium binding constant of a particular binding interaction in units of 1 / M, which is the on-rate [k on or k a ] off rate [k off or k d In some embodiments, the antigen-binding domain of the antibody or fragment thereof or CAR binds, e.g., specifically binds, at a ratio equal to 10 -5 K equal to or less than M D (i.e., the equilibrium dissociation constant of a particular binding interaction, in units of M, and the off-rate [k off or k d ] on speed [k on or k a ] (assuming a bimolecular interaction) exhibits binding affinity for a peptide epitope. For example, the equilibrium dissociation constant, KD, is 10-7 M to 10 -11 M, 10 -8 M to 10 -10 M, or 10 -9 M to 10 -10 M, etc., 10 -5 M to 10 -13 The on-rate (association rate constant; k on or k a ; units 1 / M) and off-rate (dissociation rate constant; k off or k d ; units 1 / s) can be determined using any assay method known in the art, for example, surface plasmon resonance (SPR).

[0305] In some embodiments, the binding affinity (EC 50 ) and / or the dissociation constant is from about 0.01 nM to about 500 nM, from about 0.01 nM to about 400 nM, from about 0.01 nM to about 100 nM, from about 0.01 nM to about 50 nM, from about 0.01 nM to about 10 nM, from about 0.01 nM to about 1 nM, from about 0.01 nM to about 0.1 nM, from about 0.1 nM to about 500 nM, from about 0.1 nM to about 400 nM, from about 0.1 nM to about 100 nM, from about 0.1 nM to about 50 nM, In certain embodiments, the binding affinity (EC) of the antibody to a BCMA protein, such as a human BCMA protein, is from about 0.1 nM to about 1 nM, from about 0.5 nM to about 200 nM, from about 1 nM to about 500 nM, from about 1 nM to about 100 nM, from about 1 nM to about 50 nM, from about 1 nM to about 10 nM, from about 2 nM to about 50 nM, from about 10 nM to about 500 nM, from about 10 nM to about 100 nM, from about 10 nM to about 50 nM, from about 50 nM to about 500 nM, from about 50 nM to about 100 nM, or from about 100 nM to about 500 nM. 50 ) and / or the equilibrium dissociation constant K Dis at or less than, or about: 400 nM, 300 nM, 200 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. In some embodiments, the antibody binds to a BCMA protein, such as a human BCMA protein, with a sub-nanomolar binding affinity, for example, a binding affinity of less than about 1 nM, such as about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, or about 0.1 nM or less.

[0306] In some embodiments, binding affinity may be classified as high or low affinity. In some cases, a binding molecule (e.g., an antibody or fragment thereof) or an antigen-binding domain of a CAR exhibiting low to moderate binding affinity may be 10 7 M -1 Up to 10 6 M -1 Up to 10 5 M -1 K up to A In some cases, a binding molecule (e.g., an antibody or fragment thereof) that exhibits high binding affinity for a particular epitope exhibits a binding affinity of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 K AIn some embodiments, the binding affinity (EC ) of a binding molecule, e.g., an anti-BCMA antibody or fragment thereof, or an antigen-binding domain of a CAR, to a GPRC5D protein is 50 )) and / or the equilibrium dissociation constant K D is about 0.01 nM to about 1 μM, 0.1 nM to 1 μM, 1 nM to 1 μM, 1 nM to 500 nM, 1 nM to 100 nM, 1 nM to 50 nM, 1 nM to 10 nM, 10 nM to 500 nM, 10 nM to 100 nM, 10 nM to 50 nM, 50 nM to 500 nM, 50 nM to 100 nM, or 100 nM to 500 nM. In certain embodiments, the binding affinity (EC 50 ) and / or the equilibrium dissociation constant K D is at, about, or less than about 1 μM, 500 nM, 100 nM, 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 19 nM, 18 nM, 17 nM, 16 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM or less. The degree of affinity of a particular antibody can be compared to the affinity of a known antibody, such as a reference antibody.

[0307] In some embodiments, the binding affinities of binding molecules, such as anti-BCMA antibodies or antigen-binding domains of CARs, for different antigens, e.g., BCMA proteins from different species, can be compared to determine species cross-reactivity. For example, species cross-reactivity can be classified as high cross-reactivity or low cross-reactivity. In some embodiments, the equilibrium dissociation constants K for different antigens, e.g., BCMA proteins from different species, such as human, cynomolgus monkey, or mouse, can be compared. DIn some embodiments, the species cross-reactivity of an anti-BCMA antibody or antigen-binding domain of a CAR may be high, e.g., the anti-BCMA antibody binds to human BCMA and species variant BCMA to a similar extent, e.g., the K for human BCMA D and K against species variant BCMA D In some embodiments, the species cross-reactivity of the anti-BCMA antibody or antigen-binding domain of the CAR may be low, e.g., the anti-BCMA antibody has high affinity for human BCMA but low affinity for species variant BCMA, or vice versa. For example, the K D and K against human BCMA D is 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or more, and the anti-BCMA antibody has low species cross-reactivity. The degree of species cross-reactivity may be compared to the species cross-reactivity of a known antibody, such as a reference antibody.

[0308] Some provided bispecific CARs exhibit antigen-dependent activity or signal transduction, i.e., signal transduction activity that is not measurable or at background level in the absence of antigen, such as BCMA.Thus, in some embodiments, provided CARs do not exhibit sustained signal transduction or antigen-dependent activity or signal transduction in the absence of antigen, such as BCMA, or only exhibit below background or tolerable or low levels.In some embodiments, the cells that express provided bispecific CARs exhibit biological activity or function, including cytotoxic activity, cytokine production, and proliferation ability.

[0309] In some embodiments, the biological or functional activity of a chimeric receptor, such as cytotoxic activity, can be measured using any of a number of known methods. Activity can be assessed or determined either in vitro or in vivo. In some embodiments, activity can be assessed after administration of the cells to a subject (e.g., a human). Parameters to be assessed include assessment of specific binding of engineered or natural T cells or other immune cells to an antigen, e.g., in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of engineered cells to kill target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009) and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of cells may also be measured by assaying the expression and / or secretion of certain cytokines, such as interleukin 2 (IL-2), interferon gamma (IFNγ), interleukin 4 (IL-4), tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and / or transforming growth factor beta (TGFβ). Assays for measuring cytokines are well known in the art and include, but are not limited to, ELISA, intracellular cytokine staining, cytometric bead array, RT-PCR, ELISPOT, flow cytometry, and bioassays in which cells responsive to a cytokine of interest in the presence of a test sample are tested for responsiveness. In some embodiments, biological activity is measured by assessing a clinical outcome, such as a reduction in tumor burden or burden.

[0310] In some embodiments, a reporter cell line can be employed to monitor antibody-independent activity and / or sustained signaling through cells expressing a bispecific CAR. In some embodiments, a T cell line, such as a Jurkat cell line (BCMA-negative / GPRC5D-negative), contains a reporter molecule, such as a fluorescent protein, such as red fluorescent protein, or other detectable molecule, expressed under the control of endogenous Nur77 transcriptional regulatory elements. In some embodiments, Nur77 reporter expression is cell-intrinsic and dependent on signaling through a recombinant reporter containing a primary activation signal within the T cell, a T cell receptor (TCR) component, and / or an immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling domain, such as the CD3ζ chain. Nur77 expression is generally unaffected by other signaling pathways, such as cytokine signaling or Toll-like receptor (TLR) signaling, which may operate intrinsically and may not depend on signaling through a recombinant receptor. Therefore, only cells expressing an exogenous recombinant receptor containing the appropriate signaling region, such as a bispecific CAR, can express Nur77 upon stimulation (e.g., binding of a specific antigen). In addition, in some cases, Nur77 expression can exhibit a dose-dependent response to the amount of stimulation (e.g., antigen).

[0311] In some embodiments, the provided bispecific CARs exhibit improved expression on the surface of cells, such as when compared to alternative CARs encoded by nucleotide sequences having the same amino acid sequence but without splice site removal and / or codon optimization. In some embodiments, the expression of the recombinant receptor on the surface of cells can be assessed. Approaches for determining the expression of the recombinant receptor on the surface of cells can include the use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38), Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, and monoclonal antibodies that specifically bind to the CAR polypeptide (see International Application Publication No. WO2014190273). In some embodiments, the expression of the recombinant receptor on the surface of cells, such as primary T cells, can be assessed, for example, by flow cytometry, using a binding molecule capable of binding to the recombinant receptor or a detectable portion thereof. In some embodiments, the binding molecule used to detect expression of the recombinant receptor is or comprises an anti-idiotypic antibody, e.g., an anti-idiotypic agonist antibody or portion thereof specific for a binding domain, e.g., an scFv. In some embodiments, the binding molecule is or comprises an isolated or purified antigen, e.g., a recombinantly expressed antigen.

[0312] II. Polynucleotides Encoding Recombinant Antibodies Also provided are polynucleotides encoding chimeric antigen receptors and / or portions thereof, e.g., chains. Among the polynucleotides provided are those encoding the bispecific antigen receptors (e.g., antigen-binding fragments) that bind to GPRC5D and BCMA described herein. Polynucleotides may contain natural and / or non-natural nucleotides and bases, including, for example, those with backbone modifications. The terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" may be used interchangeably and refer to polymers of nucleotides. Such nucleotide polymers may contain natural and / or non-natural nucleotides and may include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to a linear sequence of nucleotides, including a nucleic acid molecule or polynucleotide.

[0313] In some embodiments, the extracellular binding domain comprises, from amino to carboxy terminus: the V of the GPRC5D binding domain H Area and V L One of the regions, V of the GPRC5D binding domain H Area and V L The other region, V of the BCMA binding domain H Area and V L one of the regions, as well as the V of the BCMA binding domain H Area and V L In some embodiments, the extracellular binding domain comprises, from the amino to carboxy terminus, the V of the GPRC5D binding domain H Area and V L One of the regions, V of the BCMA binding domain H Area and V L One of the regions, V of the BCMA binding domain H Area and V L the other of the regions, as well as V of the GPRC5D-binding domain H Area and V LThe other of the regions. In some cases, the polynucleotide encoding the GPRC5D-binding domain contains a signal sequence encoding a signal peptide, which in some cases is encoded upstream of the nucleic acid sequence encoding the GPRC5D-binding domain or linked at the 5' end of the nucleic acid sequence encoding the GPRC5D-binding domain. In some cases, the polynucleotide containing the nucleic acid sequence encoding the GPRC5D-binding domain contains a signal sequence encoding a signal peptide. In some embodiments, the signal sequence may encode a signal peptide derived from a native polypeptide. In some embodiments, the signal sequence may encode a heterologous or non-naturally occurring signal peptide. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the IgG kappa chain set forth in SEQ ID NO:92 or encoded by the nucleotide sequence set forth in SEQ ID NOs:91 or 93-96. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the GMCSFR alpha chain set forth in SEQ ID NO:98 and encoded by the nucleotide sequence set forth in SEQ ID NO:97. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the CD8 alpha signal peptide set forth in SEQ ID NO:99. In some embodiments, non-limiting exemplary signal peptides include the signal peptide of the CD33 signal peptide set forth in SEQ ID NO: 72. In some cases, a polynucleotide encoding a GPRC5D-binding domain may contain nucleic acid sequences encoding additional molecules, such as surrogate or other markers, or may contain additional components, such as promoters, regulatory elements, and / or polycistronic elements. In some embodiments, the nucleic acid sequence encoding the GPRC5D-binding domain may be operably linked to any of the additional components.

[0314] In some embodiments, the extracellular binding domain comprises, from the amino to carboxy terminus, the V of the BCMA binding domain H Area and VL One of the regions, V of the BCMA binding domain H Area and V L The other region, V of the GPRC5D binding domain H Area and V L one of the regions, as well as V of the GPRC5D-binding domain H Area and V L In some embodiments, the extracellular binding domain comprises, from the amino to carboxy terminus, the V of the BCMA binding domain H Area and V L One of the regions, V of the GPRC5D binding domain H Area and V L One of the regions, V of the GPRC5D binding domain H Area and V L one of the regions, as well as the V of the BCMA binding domain H Area and V LThe other of the regions. In some cases, the polynucleotide encoding the BCMA binding domain contains a signal sequence encoding a signal peptide, which in some cases is encoded upstream of the nucleic acid sequence encoding the BCMA binding domain or linked at the 5' end of the nucleic acid sequence encoding the BCMA binding domain. In some cases, the polynucleotide containing the nucleic acid sequence encoding the BCMA binding domain contains a signal sequence encoding a signal peptide. In some embodiments, the signal sequence may encode a signal peptide derived from a native polypeptide. In some embodiments, the signal sequence may encode a heterologous or non-naturally occurring signal peptide. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the IgG kappa chain set forth in SEQ ID NO:92 or encoded by the nucleotide sequence set forth in SEQ ID NOs:271 or 93-96. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the GMCSFR alpha chain set forth in SEQ ID NO:98 and encoded by the nucleotide sequence set forth in SEQ ID NO:97. In some embodiments, a non-limiting exemplary signal peptide includes the signal peptide of the CD8 alpha signal peptide set forth in SEQ ID NO:99. In some embodiments, non-limiting exemplary signal peptides include the signal peptide of the CD33 signal peptide set forth in SEQ ID NO: 72. In some cases, a polynucleotide encoding a BCMA binding domain may contain nucleic acid sequences encoding additional molecules, such as surrogate or other markers, or may contain additional components, such as promoters, regulatory elements, and / or polycistronic elements. In some embodiments, the nucleic acid sequence encoding the BCMA binding domain may be operably linked to any of the additional components.

[0315] In some embodiments, a CAR provided herein is encoded by a nucleotide sequence set forth in any one of SEQ ID NOs: 105-120. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 105. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 106. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 107. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 108. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 109. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 110. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 111. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 112. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 113. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 114. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 115. In some embodiments, a CAR is encoded by a nucleotide sequence set forth in SEQ ID NO: 116. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 117. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 118. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 119. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 120.

[0316] In some embodiments, the CARs provided herein are encoded by polynucleotides containing specific features that have been optimized or designed for optimization, such as for codon usage, to reduce RNA heterogeneity, and / or to modify, e.g., increase or ensure consistency across cell production lots, expression, such as surface expression, of the encoded receptor. In some embodiments, the polynucleotide encoding the GPRC5D-binding domain or BCMA-binding domain has been modified compared to a reference polynucleotide, such as by removing cryptic or hidden splice sites, to reduce RNA heterogeneity. In some embodiments, the polynucleotide encoding the GPRC5D-binding domain or BCMA-binding domain is codon-optimized for expression in mammalian, e.g., human, cells, such as human T cells. In some embodiments, the modified polynucleotide, when expressed in a cell, results in improved expression, such as increased or more uniform or consistent levels of expression, such as surface expression. Such polynucleotides can be used in constructs for generating engineered cells that express the encoded GPRC5D-binding and BCMA-binding domains. Accordingly, also provided are cells expressing the recombinant receptors encoded by the polynucleotides provided herein and their use in adoptive cell therapy, such as for the treatment of diseases and disorders associated with GPRC5D and / or BCMA expression, e.g., multiple myeloma.

[0317] Also provided are cells, such as T cells, engineered to express a polynucleotide encoding a provided polynucleotide, including a polypeptide encoding a GPRC5D-binding domain and a BCMA-binding domain, and compositions containing such cells. In some embodiments, the polynucleotide construct is codon-optimized for expression in human cells. In some embodiments, one or more splice donor and / or acceptor sites of the polynucleotide construct are modified to reduce the heterogeneity of RNA transcribed from the construct, such as mRNA, after expression in cells.

[0318] 1. Codon optimization In some embodiments, polynucleotides are modified for expression in humans by codon optimization. In some embodiments, codon optimization can be considered before and / or after each step for splice site identification and / or splice site removal, and / or each iterative step for reducing RNA heterogeneity. Codon optimization generally involves balancing the proportion of codons selected with the abundance, e.g., published abundance, of human transfer RNA to ensure that no codons are overloaded or restricted. In some cases, such balancing is necessary or useful because most amino acids are encoded by more than one codon, and codon usage generally varies between organisms. Differences in codon usage between transfected or transduced genes or nucleic acids and between host cells can have an impact on protein expression from nucleic acid molecules. Table 2 below shows an exemplary human codon usage table. In some embodiments, codons are chosen to generate codon-optimized nucleic acid sequences by selecting codons that are balanced with their human usage. Codon redundancy for an amino acid refers to the fact that different codons code for the same amino acid, as shown in Table 2. When selecting a codon for substitution, it is desirable that the resulting mutation be a silent mutation such that the codon change does not affect the amino acid sequence. Generally, the last nucleotide (e.g., at the third position) of the codon can remain unchanged without affecting the amino acid sequence. [Table 2-1] [Table 2-2]

[0319] For example, the codons TCT, TCC, TCA, TCG, AGT, and AGC all encode serine (note that T in DNA is equivalent to U in RNA). From human codon usage, such as that shown in Table 2 above, the corresponding usage frequencies for these codons are 15.2, 17.7, 12.2, 4.4, 12.1, and 19.5, respectively. TCG corresponds to 4.4%, so if this codon were commonly used in gene production, tRNA for this codon would be limiting. In codon optimization, the goal is to balance the usage of each codon with the normal frequency of usage in the species of animal in which the transgene is to be expressed.

[0320] 2. Splice site Provided herein are polynucleotides in which one or more potential splice donor and / or splice acceptor sites have been identified, and the nucleic acid sequence at or near one or more of the identified splice donor sites has been modified. In some embodiments, the resulting modified nucleotide sequences are then synthesized and used to transduce cells and test for splicing as indicated by RNA heterogeneity.

[0321] Also provided herein are polynucleotides, such as those encoding any of the antibodies, receptors (), and / or GPRC5D-specific and / or BCMA-specific binding domains provided herein, that have been modified (such as by optimization methods) to contain one or more nucleic acid sequences found herein to reduce heterogeneity or to provide improved characteristics of a polypeptide, such as a CAR, compared to containing a different reference sequence or being unmodified. Such characteristics include improved RNA heterogeneity, such as that provided by the presence of one or more splice sites, such as one or more cryptic splice sites, and / or improved expression and / or surface expression of the encoded protein, e.g., increased, uniform, or consistent expression between cells engineered to express the polypeptide or between different therapeutic cell compositions.

[0322] Splice sites may be identified in polynucleotide sequences by collecting RNA from expressing cells, amplifying it by reverse transcription polymerase chain reaction (RT-PCR), and separating it by agarose gel electrophoresis to determine RNA heterogeneity, compared to a reference sequence. In some cases, improved sequences may be resubmitted to gene synthesis vendors for further codon optimization and splice site removal until the agarose gel RNA exhibits minimal RNA heterogeneity, followed by further cryptic splice site evaluation, modification, synthesis, and testing.

[0323] Also provided is a polynucleotide modified to remove splice sites, such as cryptic splice sites. Genomic nucleic acid sequences are generally processed in natural mammalian cells, either in conjunction with transcription or immediately after transcription, where the nascent pre-messenger ribonucleic acid (pre-RNA) transcribed from the genomic deoxyribonucleic acid (DNA) sequence is in some cases edited in a splicing manner to remove introns, and then exons are spliced in eukaryotic cells. Although consensus sequences for splice sites are known, in some embodiments, the specific nucleotide information defining a splice site may be complex or may not be readily apparent based on available methods. A cryptic splice site is a splice site that is not predicted based on a standard consensus sequence and is variably activated. Therefore, variable splicing of pre-mRNA at a cryptic splice site results in heterogeneity of the transcribed mRNA product upon expression in eukaryotic cells.

[0324] The polynucleotides generated for the expression of transgenes are typically constructed from nucleic acid sequences, such as complementary DNA (cDNA) or its parts, that do not contain introns.Therefore, splicing of such sequences is not expected to occur.Therefore, the presence of cryptic splice sites in cDNA sequences can lead to unintended or undesired splicing reactions and heterogeneity of transcribed mRNA.This heterogeneity can lead to the transcription of unintended protein products, such as truncated protein products with variable amino acid sequences that exhibit altered expression and / or activity.

[0325] In some embodiments, removal of splice sites, such as cryptic splice sites, can improve or optimize the expression of transgene products, such as polypeptides translated from transgenes, such as bispecific CAR polypeptides. Splicing at cryptic splice sites of an encoded transgene, such as an encoded CAR comprising a GPRC5D-binding domain and a BCMA-binding domain, can result in reduced protein expression, for example, expression on the cell surface, and / or reduced function, for example, reduced intracellular signaling. Provided herein are polynucleotides encoding bispecific CAR proteins that have been optimized to reduce or remove cryptic splice sites. Also provided herein are polynucleotides encoding bispecific CARs that are optimized for codon expression and / or that contain one or more sequences, such as those identified by the methods or observations herein regarding splice sites, and / or that do not contain identified splice sites, such as any of the splice sites identified herein. Some provided polynucleotides exhibit less than a certain degree of RNA heterogeneity or splice forms and / or are introduced into specific cell types, such as human T cells, such as primary human T cells, and compositions and articles of manufacture containing such polynucleotides and / or exhibiting such properties are also provided. In some embodiments, the RNA heterogeneity of the transcribed RNA is reduced by about 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more compared to a polynucleotide that has not been modified to remove cryptic splice sites and / or modified by codon optimization. In some embodiments, provided polynucleotides encoding bispecific CARs exhibit an RNA homogeneity of the transcribed RNA that is at least 70%, 75%, 80%, 85%, 90%, 95%, or more.

[0326] RNA heterogeneity can be determined by any of a number of methods provided herein, described, or known. In some embodiments, the RNA heterogeneity of a transcribed nucleic acid is determined by detecting one or more differences, such as size differences, in one or more amplification products after amplification of the transcribed nucleic acid, such as by reverse transcription polymerase chain reaction (RT-PCR). In some embodiments, RNA heterogeneity is determined based on the number of amplification products with different sizes or the proportion of various amplification products with different sizes. In some embodiments, total RNA or cytoplasmic polyadenylated RNA is collected from cells expressing the transgene to be optimized and amplified by reverse transcription polymerase chain reaction (RT-PCR) using a primer specific to the 5' untranslated region (5' UTR) and a primer specific to the 3' untranslated region (3' UTR) or a primer specific to a sequence within the transgene, where the 5' UTR-specific primer corresponds to a portion of the promoter sequence in the expression vector located upstream of the transgene in the transcribed RNA, and the 3' UTR-specific primer is located downstream of the transgene to be expressed in the transcribed RNA sequence. In certain embodiments, at least one primer complementary to a sequence in the 5' untranslated region (UTR) and at least one primer complementary to a sequence in the 3' untranslated region (UTR) are used to amplify the transgene. Those skilled in the art can separate RNA, such as messenger RNA, and analyze its heterogeneity in several ways. Non-limiting exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field-flow fractionation, and chromatography, such as size exclusion chromatography or liquid chromatography.

[0327] In some embodiments, potential cryptic splice sites (splice donor and / or acceptor sites present in a transcript, such as a transgene transcript) may result in RNA heterogeneity of that transcript after expression in a cell. In some embodiments, one or more cryptic splice sites are identified that may be present in the transgene transcript, that are undesirable and / or that may be generated in the transgene transcript from different underlying sequences after codon optimization of the transcript and / or due to mutations or transcriptional mismatches or errors. In some aspects of the provided embodiments, the splice donor site and the splice acceptor site are independently identified. In some embodiments, the splice acceptor and / or donor site are classical, non-classical, and / or cryptic splice acceptor and / or donor sites.

[0328] In some embodiments, one or more cryptic splice sites (e.g., classical, non-classical, and / or cryptic splice acceptor and / or donor sites or branch sites) in a polynucleotide, such as a polynucleotide encoding a transgene such as a recombinant receptor, that may exhibit RNA heterogeneity, are identified and / or modified. Also provided are polypeptides that have a reduced number of such splice sites compared to a reference polynucleotide.

[0329] In some embodiments, the identification of one or more splice sites in nucleic acids is an iterative process. In some embodiments, splice sites can be identified by splice site and / or codon optimization prediction tools, such as by inputting the start or reference sequence encoding a transgene, such as a bispecific CAR or the GPRC5D or BCMA binding domain contained therein, into a database, a gene synthesis vendor, or other source that can be used to identify or predict splice sites and / or to compare the start or reference sequence computationally or algorithmically for codon optimization and / or splice site removal. In some embodiments, after modifying the sequence for codon optimization and / or splice site removal, one or more further evaluations are performed on the sequence, such as the improved or modified nucleic acid sequence, and one or more other or additional splice site prediction tools are used to further evaluate splice site removal, such as cryptic splice sites.

[0330] In some embodiments, RNA heterogeneity can result from the activity of spliceosomes present in eukaryotic cells. In some embodiments, splicing typically occurs in a series of reactions catalyzed by spliceosomes. While consensus sequences for splice sites are known, in some embodiments, the specific nucleotides defining a splice site may be complex and not readily apparent based on available methods. Cryptic splice sites are splice sites that are not predicted based on standard consensus sequences and are variably activated. Thus, variable splicing at cryptic splice sites in pre-mRNAs results in heterogeneity in transcribed mRNA products after expression in eukaryotic cells. In some cases, within the intron of the spliceosome, a donor site (often located at the 5' end of the intron), a branch site (near the 3' end of the intron), and an acceptor site (located at the 3' end of the intron) are required for splicing. The splice donor site may include a GU sequence at the 5' end of the intron, accompanied by a large region that is relatively unconserved. The splice acceptor site at the 3' end of the intron may terminate in an AG sequence.

[0331] In some embodiments, splice sites, including potential cryptic splice sites, can be identified by comparing the sequence to known splice site sequences, such as those in sequence databases. In some embodiments, splice sites can be computationally identified by inputting the nucleotide sequence to be analyzed into a splice site prediction tool, such as Human Splice Finder (Desmet et al., Nucl. Acids Res. 37(9):e67 (2009)), a neural network splice site prediction tool, NNSplice (Reese et al., J. Comput. Biol., 4(4):311 (1997)), GeneSplicer (Pertea et al., Nucleic Acids Res. 2001 29(5): 1185-1190), or NetUTR (Eden and Brunak, Nucleic Acids Res. 32(3):1131 (2004)), which identifies splice sites and identifies potential splicing events at such sites. Additional splice prediction tools include RegRNA, ESEfinder, and MIT splice predictor. Splice site prediction tools such as GeneSplicer have been successfully trained and tested on databases of different species, such as humans, Drosophila melanogaster, Plasmodium, Arabidopsis, and rice. In some embodiments, different prediction tools may be adapted to different extents in different databases and / or for different species. In some embodiments, one or more prediction tools are selected based on their usefulness in a particular database and / or for a particular species. See, for example, Saxonov et al., (2000) Nucleic Acids Res., 28, 185-190.

[0332] In some embodiments, one or more splice site prediction tools are used to determine potential splice donor and / or acceptor site.In some embodiments, splice site prediction tools can be adopted that can run locally; can be retrained with user-owned data sets; can use databases for specific species (such as humans); can be compiled for multiple platforms; can enable real-time prediction for sequence selection; and / or are OSI-certified open source software that can modify specific tools or plug-ins.The exemplary tools that can be adopted include NNSplice, GeneSplicer, or both.

[0333] In some embodiments, splice site prediction tools can be used to identify a list of potential splice donor and / or splice acceptor sites within a sequence, such as a polynucleotide sequence containing a transgene sequence. In some embodiments, the prediction tool can also generate one or more prediction scores for one or more sequences in the polynucleotide, which can indicate the likelihood that one or more sequences will be splice donor or acceptor site sequences.

[0334] In some embodiments, the predicted score for a particular splice site is compared with a threshold score or reference score to determine or identify a particular splice site that is a candidate for deletion or removal. For example, in some embodiments, a predicted splice site is identified as a potential splice site when its predicted score is greater than a threshold score or reference score. In some embodiments, considerations for deleting or removing a particular splice site include the predicted score compared with the reference score or threshold score, and whether the particular splice site is desired or intentional (e.g., if the splicing event is advantageous or necessary for regulating transcription and / or translation). In some embodiments, when determining the particular donor and / or acceptor site to be deleted or removed, the possibility that the resulting splice variant will lose the desired function or have a defective function can also be considered. In some embodiments, one or more potential splice donor and / or splice acceptor sites exhibit a score of about or at least about 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 (e.g., on a scale of 1.0), for splice events or the likelihood of splice events, and the site may be a candidate for splice site deletion or removal. In some embodiments, the score for one or more potential splice donors and / or splice sites, as used, for example, by GeneSplicer, is based on the difference between the log-odds score returned by a true Markov model for the sequence and the score calculated by a false Markov model. In certain embodiments, the splice donor site and the splice acceptor site are evaluated independently or individually. In some embodiments, the splice donor site and the splice acceptor site are evaluated as a splice donor / acceptor pair.

[0335] In some embodiments, one or more splice donor and / or splice acceptor sites are removed, such as potential splice donor and / or acceptor sites that may be undesirable themselves or that may be involved in potential splicing events resulting from undesirable RNA heterogeneity. In some embodiments, removal of one or more splice sites involves altering (e.g., by substitution or replacement) one or more nucleotides within, at, containing, or near the splice donor and / or acceptor site that is a candidate for removal. In some embodiments, specific nucleotides within a codon at, containing, or near the splice site are altered (e.g., substituted or replaced). In some embodiments, this alteration (e.g., substitution or replacement) retains or preserves the amino acid encoded by the specific codon at the site while simultaneously removing the potential splice donor and / or acceptor site.

[0336] In some embodiments, the codons at or near a splice site that are subject to modification include one or more codons with one or both of the two nucleotides of a potential splice site (in some cases referred to as "splice site codons"). If potential splicing is predicted to occur between two nucleotides within a codon, only this codon is the splice site codon for that splice site. If potential splicing occurs between two adjacent codons, for example, between the last nucleotide of a first codon and the first nucleotide of the next codon, the two codons are splice site codons. For example, for a splice site predicted to be at the boundary of two codons, the two adjacent codons can be candidates for nucleotide modification. In some embodiments, one or more codons include one splice site codon. In some embodiments, one or more codons include both splice site codons. In some embodiments, a potential splice donor site is removed by modifying one or both splice site codons. In some embodiments, potential splice acceptor donor sites are eliminated by modifying one or both splice site codons. In some embodiments, one or both codons at a splice site are left unmodified, for example, if there are no synonymous codons for that splice site codon. In some embodiments, if there are no synonymous codons available for a particular splice site codon, one or more nucleotides of a nearby codon may be modified. In some embodiments, the one or more codons that are modified include a splice site codon, where the modification involves changing one or both nucleotides at the splice site to a different nucleotide or nucleotides. In some embodiments, a splice donor site is eliminated by modifying one or more splice site codons, where the modification does not change one or two of the nucleotides at the splice site, but modifies nearby nucleotides, for example, some of the codons adjacent to the splice site.In some embodiments, nearby or adjacent nucleotides that may be modified include modifying nucleotides that are part of nearby or adjacent codons, such as codons within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 codons upstream or downstream of the splice site codon.

[0337] In some cases, predicted splice sites can be manually modified to reduce the likelihood of splicing while preserving the encoded amino acids. In some embodiments, one or more predicted splice sites with at least 80%, 85%, 90%, or 95% splice site likelihood are manually modified to reduce the likelihood of splicing events. In some embodiments, one or more modifications are nucleotide replacements or substitutions of 1, 2, 3, 4, 5, 6, or 7 nucleotides. In some embodiments, the modifications are at the splice donor site junction or the splice acceptor site junction. In some embodiments, at least one or more nucleotide modifications are within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues of the splice site junction of the splice acceptor and / or splice donor site. In some embodiments, a library of modified nucleic acid sequences that reduces the likelihood of cryptic splice sites can be refined. In some embodiments, splice donor sites and splice acceptor sites are evaluated as a splice donor / acceptor pair. In certain embodiments, splice donor sites and splice acceptor sites are evaluated independently or individually, and are not evaluated as part of a splice donor / acceptor pair. In some embodiments, one or more predicted splice sites are not removed. In some embodiments, splice sites, such as known or predicted splice sites within the promoter region of a transcript, are not removed.

[0338] In some embodiments, one or more potential donor splice sites are removed by modifying one or two splice site codons, or one or more nearby or adjacent codons (e.g., if synonymous codons are not available for the splice site). In some embodiments, one or more potential acceptor splice sites are removed by modifying one or two splice site codons, or one or more nearby or adjacent codons (e.g., if synonymous codons are not available for the splice site). In some embodiments, the nearby or adjacent codons to be modified include codons within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 codons upstream or downstream of the splice site codon, for example, within 1, 2, or 3 codons from the splice site. In some embodiments, potential splicing branch sites are removed or deleted. In some embodiments, nucleotides within a codon at or near the branch site can be modified to eliminate potential splicing and / or reduce RNA heterogeneity. In some embodiments, the modification of one or more nucleotides may involve the substitution or replacement of one of the nucleotides potentially involved in splicing (such as those at the splice donor site, splice acceptor site, or splice branch site), such that the amino acid encoded by the codon is preserved and the substitution or replacement of the nucleotide does not change the polynucleotide sequence encoded by the polynucleotide. In some cases, the third position of a codon is more degenerate than the other two positions. Thus, various synonymous codons may encode a particular amino acid (see, e.g., Section II above). In some embodiments, the modification involves replacing a codon with a synonymous codon used in the species (e.g., human) of the cell into which the polynucleotide is introduced. In some embodiments, the species is human.In some embodiments, one or more codons are replaced with the corresponding synonymous codon that is most frequently used in the species, or with a synonymous codon that has a similar frequency of usage (e.g., closest frequency of usage) to the corresponding codon (see, e.g., Section II.1 above).

[0339] In some embodiments, after the initial proposed modifications, the transgene's candidate requirement for splice site removal is evaluated. In some embodiments, the proposed modifications can be reevaluated after modification and / or codon optimization to evaluate the proposed modifications and identify any additional potential splice sites. In some embodiments, after modifying the sequence for codon optimization and / or splice site removal, one or more further evaluations of the sequence, such as the improved or modified nucleic acid sequence, are performed using the same or one or more other or additional splice prediction tools to further evaluate for removal of splice sites, such as cryptic splice sites. In some embodiments, the proposed modifications are considered in subsequent steps, and iterative optimization can be used. In some embodiments, either method of identification and / or modification steps can be repeated, for example, until the heterogeneity of the transcript is reduced compared to the initially determined heterogeneity of the transcript. In some embodiments, additional or different modifications, such as different nucleotide substitutions at the same codon or modifications at different positions or codons, can be made after the iterative evaluation and evaluation. In some embodiments, a corresponding different synonymous codon may be used, such as the second most frequently used in a particular species, or a codon with a similar frequency of use to the corresponding codon (see, e.g., Section II.1 above).

[0340] In some embodiments, the proposed modification can be further evaluated, for example, to assess whether the modification creates an undesired or additional restriction site within the polynucleotide. In some embodiments, the additional restriction site may be undesirable, and further or different modifications (e.g., a different nucleotide substitution in the same codon, or a modification at a different position or codon) can be considered. In some embodiments, a specific restriction site, such as a designated restriction site, is avoided. In some embodiments, if the modification does not substantially reduce the splice site prediction score, additional or alternative modifications can be proposed. In some embodiments, the splice site prediction score can be reduced or decreased by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% after one or more iterations of the method.

[0341] In some embodiments, a computer system may be implemented to perform one or more steps, tools, functions, processes, or scripts. In some embodiments, splice site prediction, evaluation, and modification for splice site deletion or removal may be performed by a computer-implemented method and / or a method including steps that are computer-implemented steps. In some embodiments, any one of the steps of comparing sequences to known databases, calculating splice site prediction scores, determining potential nucleotide modifications, codon optimization, and / or iteratively may be computer-implemented or performed using computer-implemented steps, tools, functions, processes, or scripts. In certain embodiments, a computer system is provided that includes a processor and memory, wherein the memory stores instructions operable to cause the processor to perform any one or more steps of the methods provided herein. In some embodiments, the steps, functions, processes, or scripts are performed computationally, e.g., using one or more computer programs and / or through the use of computational algorithms.

[0342] Exemplary steps, functions, processes, or scripts for identifying and / or removing potential splice sites include one or more of the following: selecting a sequence, writing a FASTA-formatted sequence, loading a codon table (e.g., from www.kazusa.or.jp / codon), launching GeneSplicer, loading predictions, analyzing codons, determining overlaps in predictions, identifying the next most frequently used synonymous codon, verifying restriction sites, generating annotations, or evaluating other codons. Certain steps may evaluate both the forward and reverse strands. In some embodiments, previously annotated splice site modifications are also considered to enable iterative optimization. In some embodiments, any one or more of the steps, functions, processes, or scripts may be repeated.

[0343] In some embodiments, the provided polynucleotides encoding the CARs provided herein or constructs provided herein contain modifications to remove one or more splice donor and / or acceptor sites that may contribute to splice events and / or reduced expression and / or increased RNA heterogeneity. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 119. In some embodiments, the CAR is encoded by the nucleotide sequence set forth in SEQ ID NO: 120.

[0344] 3. Other features Also provided are vectors containing the polynucleotides, for example, for producing chimeric antigen receptors, and host cells containing the vectors. Also provided are methods for producing chimeric antigen receptors. The nucleic acid may encode a chimeric antigen receptor comprising the VL and / or VH regions of an antibody (e.g., the antibody's light and / or heavy chains). The nucleic acid may encode one or more amino acid-binding domains (e.g., a BCMA-binding domain and a GPRC5D-binding domain), each comprising the VL and / or VH regions of an antibody (e.g., the antibody's light and / or heavy chains). In further embodiments, one or more vectors (e.g., expression vectors) containing such polynucleotides are provided. In further embodiments, host cells containing such polynucleotides are provided. In one such embodiment, the host cell contains a vector (e.g., is transduced with a vector) comprising a nucleic acid encoding a chimeric antigen receptor comprising an antibody's VH region. In another such embodiment, the host cell contains (e.g., is transduced with) (1) a vector containing a nucleic acid encoding a chimeric antigen receptor comprising an antibody VL region and an antibody VH region, or (2) a vector containing a nucleic acid encoding a chimeric antigen receptor comprising a first antibody and a second antibody. In some embodiments, the host cell contains (e.g., is transduced with) one or more vectors containing one or more nucleic acids encoding one or more chimeric antigen receptors. In some embodiments, one or more such host cells are provided. In some embodiments, compositions containing one or more such host cells are provided. In some embodiments, the one or more host cells can express different chimeric antigen receptors or the same chimeric antigen receptor. In some embodiments, each of the host cells can express more than one chimeric antigen receptor.

[0345] Also provided are methods for making bispecific chimeric antigen receptors that bind to BCMA and GPRC5D. For recombinant production of chimeric receptors, nucleic acid sequences encoding, for example, a chimeric receptor antibody as described herein, may be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid sequences may be readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). In some embodiments, methods for making bispecific chimeric antigen receptors are provided, comprising culturing host cells containing nucleic acid sequences encoding the antibodies provided above (i.e., the BCMA-binding domain and the GPRC5D-binding domain) under conditions suitable for receptor expression.

[0346] In some embodiments, methods are provided for making cell compositions comprising cells that express bispecific chimeric antigen receptors.

[0347] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi and yeast are suitable hosts for cloning or expression of antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been modified to mimic or approximate that of human cells, resulting in the production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0348] Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13, and FUT8 CHO cells; PER.C6® cells; and NSO cells. In some embodiments, antibody heavy and / or light chains (e.g., VH and / or VL regions) may be expressed in yeast (see, e.g., U.S. Application Publication No. US 2006 / 0270045 A1). In some embodiments, particular eukaryotic host cells are selected based on their ability to confer desired post-translational modifications to heavy and / or light chains (e.g., VH and / or VL regions). For example, in some embodiments, CHO cells produce polypeptides with higher levels of sialylation than the same polypeptides produced in 293 cells. In certain examples, immune cells, such as human immune cells, are used to express the provided polypeptides encoding chimeric antigen receptors. In some examples, the immune cells are T cells, such as CD4+ and / or CD8+ immune cells.

[0349] III. Engineered Cells Also provided are cells, such as engineered cells containing recombinant receptors (e.g., chimeric antigen receptors), such as those containing an extracellular binding domain that includes both the GPRC5D-binding domain and the BCMA-binding domain provided herein. Also provided are populations of such cells, compositions containing and / or enriched for such cells, e.g., wherein cells expressing the GPRC5D-binding domain and the BCMA-binding domain represent at least 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent, or more of all cells in the composition, or of a particular type of cell, such as T cells, CD8+ cells, or CD4+ cells.

[0350] Also provided are cells, such as engineered cells, engineered to contain a recombinant receptor (e.g., a CAR) comprising a GPRC5D binding domain and a BCMA binding domain. In some embodiments, the recombinant receptor is a tandem CAR comprising a GPRC5D binding domain and a BCMA binding domain. The GPRC5D binding domain can be any known GPRC5D binding domain, such as those contained in the anti-GPRC5D CARs described herein or elsewhere (see, for example, WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245). Exemplary GPRC5D binding domains are described in Section I. The BCMA binding domain may be any known BCMA binding domain, such as those contained in the anti-BCMA CARs described herein or elsewhere (see, for example, WO2013 / 154760, WO2015 / 052538, WO2015 / 090229, WO2015 / 092024, WO2015 / 158671, WO2016 / 014565, WO2016 / 014789, WO2016 / 094304, WO2016 / 166630, WO2017 / 021450, WO2017 / 083511, WO2017 / 130223, WO2017 / 211900, WO2018 / 085690, WO2018 / 028647). Exemplary BCMA binding domains are described in Section I.

[0351] In some embodiments, the engineered cells provided herein can be combined with one or more engineered cell populations that express one or more other recombinant receptors.These engineered cell populations can be formulated into the same or separate compositions.Compositions include pharmaceutical compositions and formulations for administration, such as those for adoptive cell therapy.Also provided is a therapeutic method for administering any of the cells or compositions provided herein to a subject, for example, a patient.

[0352] Therefore, also provided are genetically engineered cells that express a recombinant receptor containing an antibody, for example, a cell containing a CAR. The cells are generally eukaryotic cells, such as mammalian cells, and typically human cells. In some embodiments, the cells are derived from blood, bone marrow, lymph, or lymphoid organs, and are cells of the immune system, such as myeloid or lymphoid cells, including cells of innate or adaptive immunity, such as lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells include T cells. In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., total T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, proliferation, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, the cells include CD4+ T cells. In some embodiments, the cells include CD8+ T cells. In some embodiments, the cells include CD4+ and CD8+ T cells. The cells may be allogeneic and / or autologous with respect to the subject being treated. Methods include existing methods. In some embodiments, such as those relating to existing technology, the cells are multipotent and / or pluripotent, e.g., stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from a subject, preparing, treating, culturing, and / or manipulating them as described herein, and reintroducing them into the same patient, before or after cryopreservation.

[0353] Subpopulations of T cells, and / or CD4+ and / or CD8+ T cells, include naive T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, natural and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0354] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0355] In some embodiments, the cells contain one or more polynucleotides introduced via genetic engineering, thereby expressing recombinant or genetically engineered products of such polynucleotides. In some embodiments, the polynucleotides are heterologous, i.e., not normally present in the cell or sample obtained from the cell, such as those obtained from another organism or cell, e.g., not normally found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the polynucleotides are non-naturally occurring, such as polynucleotides not found in nature, including those that contain chimeric combinations of polypeptides encoding various domains from multiple different cell types. In some embodiments, the cells (e.g., engineered cells) contain a vector (e.g., a viral vector, an expression vector, etc.) described herein, such as a vector comprising a nucleic acid encoding a recombinant receptor described herein.

[0356] A. Vectors and Methods for Genetic Manipulation Also provided are methods, polynucleotides, compositions, and kits for expressing bispecific recombinant receptors (e.g., CARs) and for producing genetically engineered cells that express such receptors. In some embodiments, one or more recombinant receptors (e.g., CARs) can be genetically engineered into a cell or multiple cells. Genetic engineering generally involves introducing nucleic acids encoding recombinant or engineered components into cells, such as by lentiviral transduction, retroviral transduction, transfection, or transformation.

[0357] In some embodiments, gene transfer is achieved by first stimulating the cells, such as by combining them with a stimulus that induces a response such as proliferation, survival, and / or activation, e.g., as measured by expression of cytokines or activation markers, then transducing the activated cells and expanding them in culture to sufficient numbers for clinical application.

[0358] In some contexts, overexpression of a stimulatory factor (e.g., a lymphokine or cytokine) can be toxic to a subject. Thus, in some contexts, engineered cells contain gene segments that make the cells susceptible to negative selection in vivo, such as upon administration in adoptive immunotherapy. For example, in some embodiments, cells can be engineered so that they can be eliminated as a result of changes in the in vivo conditions of the patient to whom they are administered. The negatively selectable phenotype may be due to the insertion of a gene that confers sensitivity to an administered substance, e.g., a compound. Negatively selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Wigler et al., Cell 2:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HRPT) gene, the cellular adenosine phosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0359] In some embodiments, cells are further engineered to promote the expression of cytokines or other factors.Various methods for the introduction of genetically engineered components, such as antigen receptors, such as CAR, are well known and can be used with the provided methods and compositions.Exemplary methods include those for transferring the polynucleotide encoding receptor, including those via virus, such as retrovirus or lentivirus, transduction, transposon, and electroporation.

[0360] In some embodiments, recombinant polynucleotides are transferred into cells using recombinant infectious viral particles, such as vectors derived from Simian Virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, the recombinant polynucleotide is transferred into T cells using a recombinant lentiviral vector (lentivector; see, e.g., Amado et al., Science. 1999 Jul 30;285(5428):674-676), such as an HIV-1 lentiviral-based vector, or a retroviral vector, such as a gammaretroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557).

[0361] In some embodiments, the retroviral or lentiviral vector has long terminal repeats (LTRs). In some embodiments, the vector is derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen focus-forming virus (SFFV), human immunodeficiency virus type 1 (HIV-1), human immunodeficiency virus type 2 (HIV-2 / SIV), or adeno-associated virus (AAV). In some embodiments, the vector is a self-inactivating (SIN) vector. In some embodiments, the vector is a conditionally replicating (mobilizing) vector. Most lentiviral vectors are derived from human, feline, or simian lentiviruses. Most retroviral vectors are derived from murine retroviruses. In some embodiments, the lentivirus or retrovirus, including those derived from any avian or mammalian cell source, is typically amphotropic, meaning that it can infect multiple host cells, including humans. In some embodiments, the gene to be expressed replaces the gag, pol, and / or env sequences of the retrovirus. Methods for lentiviral transduction are known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505.A number of exemplary retroviral systems have also been described (e.g., Amado et al., (1999) Science 285(5428):674-676, US Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0362] In some embodiments, recombinant polynucleotides are transferred into T cells via electroporation (see, e.g., Chicaybam et al. (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant polynucleotides are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-promoted biolistics (Johnston (1990) Nature 346: 776-777); and strontium phosphate DNA co-precipitation (Brash et al., (1987) Mol. Cell Biol. 7: 2031-2034). Approaches and vectors for the transfer of polynucleotides encoding recombinant products are described, for example, in International Patent Application Publication No. WO2014055668 and U.S. Patent No. 7,446,190.

[0363] Additional polynucleotides, e.g., genes, for introduction include those that improve the outcome of treatment, such as by promoting the survival and / or function of transferred cells; genes that provide genetic markers for cell selection and / or evaluation, such as those for assessing in vivo survival or localization; and genes that improve safety by making cells susceptible to negative selection in vivo, as described, for example, in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991) and Riddell et al., Human Gene Therapy 3:319-338 (1992). See also publications PCT / US91 / 08442 and PCT / US94 / 05601 by Lupton et al., which describe the use of bifunctional selectable fusion genes derived by fusing a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al., U.S. Pat. No. 6,040,177, columns 14-17.

[0364] In some embodiments, one or more recombinant receptors (e.g., CARs) can be genetically engineered to be expressed in a cell or a plurality of cells. In some embodiments, the recombinant receptor is a CAR. In some embodiments, the CAR comprises two antigen-binding domains. In some embodiments, the CAR comprises a GPRC5D-binding domain that binds to GPRC5D (e.g., human GPRC5D) and a BCMA-binding domain that binds to BCMA (e.g., human BCMA). In some embodiments, the GPRC5D-binding domain and the BCMA-binding domain of the CAR are separated by a linker, such as a polypeptide linker.

[0365] In some embodiments, the vector or construct may contain a promoter and / or enhancer or regulatory element for regulating the expression of the encoded recombinant receptor. The promoter and / or enhancer or regulatory element may be a conditional promoter, enhancer, and / or regulatory element. In some examples, these elements drive the expression of the transgene. In some examples, the CAR transgene may be operably linked to a promoter such as the EF1 alpha promoter (SEQ ID NO: 61) with the HTLV1 enhancer. In some embodiments, the CAR transgene is operably linked to a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE; SEQ ID NO: 62) located downstream of the transgene.

[0366] In some embodiments, a vector or construct may contain a single promoter driving the expression of one or more nucleic acid molecules. In some embodiments, such nucleic acid molecules, e.g., transcripts, may be polycistronic (dicistronic or tricistronic, see, e.g., U.S. Pat. No. 6,060,273). For example, in some embodiments, a transcription unit may be engineered to be a bicistronic unit containing an IRES (internal ribosome entry site), which allows for co-expression of gene products (e.g., first and second chimeric receptors) by messages from a single promoter.

[0367] Alternatively, in some cases, a single promoter may direct the expression of a single open reading frame (ORF), two or three genes (e.g., encoding first and second molecules, such as antibody recombinant receptors) separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A cleavage sequence), or RNA containing a protease recognition site (e.g., furin). Thus, the ORF encodes a single polypeptide, which is cleaved into individual proteins during (in the case of T2A) or after translation. In some cases, a peptide such as T2A can cause the ribosome to skip synthesis of the peptide bond at the C-terminus of the 2A element (ribosomal skip), which causes cleavage between the 2A sequence and the next downstream peptide (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and polynucleotides disclosed herein include, but are not limited to, the 2A sequences of foot-and-mouth disease virus (F2A; e.g., SEQ ID NO: 63 or 64), equine rhinovirus A (E2A; e.g., SEQ ID NO: 65 or 66), Thosea asigna virus (T2A, e.g., SEQ ID NO: 67, 68, or 69), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 70 or 71), as described in U.S. Patent Application Publication No. 20070116690. In some embodiments, one or more different or separate promoters drive the expression of one or more nucleic acid molecules encoding one or more binding molecules, e.g., recombinant receptors.

[0368] Any of the recombinant receptors provided herein, for example, a bispecific CAR that binds to GPRC5D and BCMA, can be encoded by a polynucleotide containing one or more nucleic acid molecules encoding the receptor in any combination or arrangement. For example, one, two, three, or more polynucleotides can encode one, two, three, or more different receptors or domains. In some embodiments, one vector or construct contains nucleic acid molecules encoding one or more recombinant receptors, and another vector or construct contains nucleic acid molecules encoding additional binding molecules, such as antibodies and / or recombinant receptors. Each of the nucleic acid molecules can also encode one or more surrogate markers, such as fluorescent proteins (e.g., green fluorescent protein (GFP)) or cell surface markers (e.g., surface cleavage markers such as cleaved EGFR (tEGFR)), which can be used to confirm that cells have been transduced or engineered to express the receptor. For example, in some embodiments, an exogenous marker gene is utilized in combination with treatment with engineered cells to enable cell detection or selection, and in some cases, to promote cell death by ADCC. Exemplary marker genes include truncated epidermal growth factor receptor (EGFRt), which can be co-expressed in transduced cells with a transgene of interest (e.g., a CAR or TCR) (see, e.g., U.S. Pat. No. 8,802,374). EGFRt contains an epitope recognized by the antibody cetuximab (Erbitux®). For this reason, Erbitux® can be used to identify or select cells engineered with an EGFRt construct, including cells co-engineered with another recombinant receptor, such as a chimeric antigen receptor (CAR).

[0369] In some embodiments, the marker is a molecule, eg, a cell surface protein, or portion thereof, that is not naturally found on T cells or that is not naturally found on the surface of T cells.

[0370] In some embodiments, the molecule is a non-self molecule, e.g., a non-self protein, i.e., one that is not recognized as "self" by the immune system of the host into which the cells will be adoptively transferred.

[0371] In some embodiments, the marker does not serve a therapeutic function and serves no effect other than its use as a marker for genetic manipulation, e.g., to select successfully manipulated cells, etc. In some embodiments, the marker may be a therapeutic molecule or other molecule that exerts a desired effect, such as a ligand encountered by the cells in vivo, such as a costimulatory or immune checkpoint molecule to enhance and / or attenuate the response of the cells upon adoptive transfer and encounter with the ligand.

[0372] Also provided are compositions containing one or more of the nucleic acid molecules, vectors, or constructs, such as any of those described above. In some embodiments, the nucleic acid molecules, vectors, constructs, or compositions can be used to engineer cells, such as T cells, to express any of the binding molecules, e.g., antibodies or recombinant receptors, and / or additional binding molecules.

[0373] B. Preparation of Cells for Manipulation In some embodiments, preparation of engineered cells involves one or more culture and / or preparation steps. Cells for introducing a recombinant receptor (e.g., a CAR) may be isolated from a sample, such as a biological sample, obtained from or derived from a subject. In some embodiments, the subject from whom the cells are isolated has a disease or condition or is in need of cell therapy or will be provided with cell therapy. In some embodiments, the subject is a human in need of a particular therapeutic intervention, such as adoptive cell therapy, for which cells are isolated, treated, and / or engineered.

[0374] Thus, in some embodiments, the cells are primary cells, such as primary human T cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or processed samples. Biological samples include, but are not limited to, bodily fluids such as blood, plasma, serum, cerebrospinal fluid, body fluids, synovial fluid, urine, and sweat, tissue, and organ samples (including samples processed therefrom).

[0375] In some embodiments, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or apheresis or leukapheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom. Samples include samples from autologous and allogeneic sources in the context of cell therapy, such as adoptive cell therapy.

[0376] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. The cells, in some embodiments, are obtained from a heterologous source, e.g., mouse, rat, non-human primate, or pig.

[0377] In some embodiments, cell isolation involves one or more preparation steps and / or non-affinity-based cell separation steps. In some instances, cells are washed, centrifuged, and / or incubated in the presence of one or...

Claims

1. (a) an extracellular domain comprising a GPRC5D-binding domain that comprises a heavy chain variable (VH) region and a light chain variable (VL) region and that binds to GPRC5D, and a BCMA-binding domain that comprises a VH region and a VL region and that binds to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus: (i) one of the VH and VL regions of the GPRC5D-binding domain, one of the VH and VL regions of the BCMA-binding domain, the other of the VH and VL regions of the BCMA-binding domain, and the other of the VH and VL regions of the GPRC5D-binding domain; or (ii) comprises one of the VH and VL regions of the BCMA-binding domain, one of the VH and VL regions of the GPRC5D-binding domain, the other of the VH and VL regions of the GPRC5D-binding domain, and the other of the VH and VL regions of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

2. The bispecific CAR according to claim 1, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, one of the VH region and the VL region of the GPRC5D-binding domain, one of the VH region and the VL region of the BCMA-binding domain, the other of the VH region and the VL region of the BCMA-binding domain, and the other of the VH region and the VL region of the GPRC5D-binding domain.

3. The bispecific CAR according to claim 1 or 2, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VH region of the GPRC5D-binding domain, a VH region of the BCMA-binding domain, a VL region of the BCMA-binding domain, and a VL region of the GPRC5D-binding domain.

4. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the GPRC5D-binding domain, the VH region of the BCMA-binding domain, the VL region of the BCMA-binding domain, and the VL region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

5. The bispecific CAR according to claim 1 or 2, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VH region of the GPRC5D-binding domain, a VL region of the BCMA-binding domain, a VH region of the BCMA-binding domain, and a VL region of the GPRC5D-binding domain.

6. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the GPRC5D-binding domain, the VL region of the BCMA-binding domain, the VH region of the BCMA-binding domain, and the VL region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

7. The bispecific CAR according to claim 1 or 2, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VL region of the GPRC5D-binding domain, a VH region of the BCMA-binding domain, a VL region of the BCMA-binding domain, and a VH region of the GPRC5D-binding domain.

8. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the GPRC5D-binding domain, the VH region of the BCMA-binding domain, the VL region of the BCMA-binding domain, and the VH region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

9. The bispecific CAR according to claim 1 or 2, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VL region of the GPRC5D-binding domain, a VL region of the BCMA-binding domain, a VH region of the BCMA-binding domain, and a VH region of the GPRC5D-binding domain.

10. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the GPRC5D-binding domain, the VL region of the BCMA-binding domain, the VH region of the BCMA-binding domain, and the VH region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

11. The bispecific CAR according to claim 1, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, one of the VH region and the VL region of the BCMA-binding domain, one of the VH region and the VL region of the GPRC5D-binding domain, the other of the VH region and the VL region of the GPRC5D-binding domain, and the other of the VH region and the VL region of the BCMA-binding domain.

12. The bispecific CAR according to claim 1 or 11, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VH region of the BCMA-binding domain, a VH region of the GPRC5D-binding domain, a VL region of the GPRC5D-binding domain, and a VL region of the BCMA-binding domain.

13. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the BCMA-binding domain, the VH region of the GPRC5D-binding domain, the VL region of the GPRC5D-binding domain, and the VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

14. The bispecific CAR according to claim 1 or 11, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VH region of the BCMA-binding domain, a VL region of the GPRC5D-binding domain, a VH region of the GPRC5D-binding domain, and a VL region of the BCMA-binding domain.

15. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the BCMA-binding domain, the VL region of the GPRC5D-binding domain, the VH region of the GPRC5D-binding domain, and the VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

16. The bispecific CAR according to claim 1 or 11, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VL region of the BCMA-binding domain, a VH region of the GPRC5D-binding domain, a VL region of the GPRC5D-binding domain, and a VH region of the BCMA-binding domain.

17. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the BCMA-binding domain, the VH region of the GPRC5D-binding domain, the VL region of the GPRC5D-binding domain, and the VH region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor comprising:

18. The bispecific CAR according to claim 1 or 11, wherein the extracellular domain comprises, in order from the amino terminus to the carboxy terminus, a VL region of the BCMA-binding domain, a VL region of the GPRC5D-binding domain, a VH region of the GPRC5D-binding domain, and a VH region of the BCMA-binding domain.

19. (a) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the BCMA-binding domain, the VL region of the GPRC5D-binding domain, the VH region of the GPRC5D-binding domain, and the VH region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

20. The bispecific CAR according to any one of claims 1 to 19, wherein (a) the VH region or VL region of the GPRC5D-binding domain; and (b) the VH region or VL region of the BCMA-binding domain are linked via a linker.

21. The bispecific CAR of claim 20, wherein the linker is a flexible peptide linker.

22. The bispecific CAR of claim 20 or 21, wherein the linker is 4 to 12 amino acids in length.

23. The bispecific CAR according to any one of claims 20 to 22, wherein the linker is or comprises the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO:

22.

24. (a) the VH and VL regions of the GPRC5D binding domain are linked by a linker; or (b) the VH and VL regions of the BCMA binding domain are linked by a linker; The bispecific CAR according to any one of claims 1 to 23.

25. The bispecific CAR according to claim 24, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 17 or SEQ ID NO:

18.

26. (a) an extracellular domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and a GPRC5D-binding domain that binds to GPRC5D, and a VH region and a VL region and a BCMA-binding domain that binds to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus: (i) the VH region of the GPRC5D binding domain; (ii) a linker shown in SEQ ID NO: 21; (iii) the VL region of the BCMA-binding domain; (iv) a linker shown in SEQ ID NO: 17; (v) the VH region of the BCMA-binding domain; (vi) a linker as set forth in SEQ ID NO: 21, and (vii) comprising a VL region of a GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

27. (a) (i) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and (ii) a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, one of the VH region and the VL region of the BCMA-binding domain, the other of the VH region and the VL region of the BCMA-binding domain, one of the VH region and the VL region of the GPRC5D-binding domain, and the other of the VH region and the VL region of the GPRC5D-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

28. (a) (i) an extracellular domain comprising a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and (ii) a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VL region of the GPRC5D-binding domain, the VH region of the GPRC5D-binding domain, one of the VH region and VL region of the BCMA-binding domain, and the other of the VH region and VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) intracellular signaling domain A bispecific chimeric antigen receptor (CAR), comprising:

29. The bispecific CAR according to claim 27 or 28, wherein the GPRC5D-binding region and the BCMA-binding region are linked by a linker.

30. The bispecific CAR of claim 29, wherein the linker is a flexible peptide linker.

31. The bispecific CAR of claim 29 or 30, wherein the linker is 4 to 12 amino acids in length.

32. The bispecific CAR according to any one of claims 29 to 31, wherein the linker comprises the amino acid sequence shown in SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO:

24.

33. The bispecific CAR according to any one of claims 27 to 32, wherein the VH region and VL region of the BCMA-binding domain are linked by a linker comprising the amino acid sequence shown in SEQ ID NO:

17.

34. (a) an extracellular domain comprising (i) a GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and binding to GPRC5D, and (ii) a BCMA-binding domain comprising a VH region and a VL region and binding to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus, the VH region of the GPRC5D-binding domain, the VL region of the GPRC5D-binding domain, one of the VH region and VL region of the BCMA-binding domain, and the other of the VH region and VL region of the BCMA-binding domain; (b) a spacer; (c) a transmembrane domain; and (d) comprises an intracellular signaling domain; wherein the GPRC5D binding domain and the BCMA binding domain are linked by a linker comprising the sequence shown in SEQ ID NO: 19 or SEQ ID NO: 21; Bispecific chimeric antigen receptors (CARs).

35. The bispecific CAR according to any one of claims 1 to 34, wherein the VH region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

36. The bispecific CAR according to any one of claims 1 to 35, wherein the VL region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

37. The bispecific CAR according to any one of claims 1 to 36, wherein the VH region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3, respectively; and the VL region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.

38. The bispecific CAR according to any one of claims 1 to 37, wherein the VH region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

7.

39. The bispecific CAR according to any one of claims 1 to 38, wherein the VL region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

8.

40. The bispecific CAR according to any one of claims 1 to 39, wherein the VH region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 7; and the VL region of the GPRC5D-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

8.

41. The bispecific CAR according to any one of claims 1 to 40, wherein the VH region of the GPRC5D-binding domain comprises the amino acid sequence shown in SEQ ID NO:

7.

42. The bispecific CAR according to any one of claims 1 to 41, wherein the VL region of the GPRC5D-binding domain comprises the amino acid sequence shown in SEQ ID NO:

8.

43. The bispecific CAR according to any one of claims 1 to 42, wherein the VH region of the GPRC5D-binding domain comprises the amino acid sequence shown in SEQ ID NO: 7; and the VL region of the GPRC5D-binding domain comprises the amino acid sequence shown in SEQ ID NO:

8.

44. The bispecific CAR according to any one of claims 1 to 43, wherein the VH region of the BCMA-binding domain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

45. The bispecific CAR according to any one of claims 1 to 44, wherein the VL region of the BCMA-binding domain comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

46. The bispecific CAR according to any one of claims 1 to 45, wherein the VH region of the BCMA-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

15.

47. The bispecific CAR according to any one of claims 1 to 46, wherein the VL region of the BCMA-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

16.

48. The bispecific CAR according to any one of claims 1 to 47, wherein the VH region of the BCMA-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO: 15; and the VL region of the BCMA-binding domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:

16.

49. The bispecific CAR according to any one of claims 1 to 48, wherein the VH region of the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO:

15.

50. The bispecific CAR according to any one of claims 1 to 49, wherein the VL region of the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO:

16.

51. The bispecific CAR according to any one of claims 1 to 50, wherein the VH region of the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO: 15; and the VL region of the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO:

16.

52. The bispecific CAR according to any one of claims 1, 20 to 25, and 35 to 51, wherein the extracellular binding domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 77, 78, 79, and 80.

53. The bispecific CAR according to any one of claims 1, 20 to 25, and 35 to 51, wherein the extracellular binding domain comprises an amino acid sequence set forth in SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90.

54. The bispecific CAR according to any one of claims 1, 2, 5, 6, 20 to 26, 35 to 51, and 53, wherein the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:

83.

55. The bispecific CAR according to any one of claims 1, 2, 7, 8, 20 to 25, 35 to 51, and 53, wherein the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:

84.

56. The bispecific CAR according to any one of claims 1, 2, 5, 6, 20 to 25, 35 to 51, and 53, wherein the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:

87.

57. The bispecific CAR according to any one of claims 1, 11, 14, 15, 20 to 25, 35 to 51, and 53, wherein the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:

81.

58. The bispecific CAR according to any one of claims 1, 11, 16, 17, 20 to 25, 35 to 51, and 53, wherein the extracellular binding domain comprises the amino acid sequence of SEQ ID NO:

85.

59. The bispecific CAR according to any one of claims 1, 11, 18 to 25, 35 to 51, and 53, wherein the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:

86.

60. The bispecific CAR according to any one of claims 1, 11, 16, 17, 20 to 25, 35 to 51, and 53, wherein the extracellular binding domain comprises the amino acid sequence of SEQ ID NO:

90.

61. The bispecific CAR according to any one of claims 1 to 60, wherein the spacer comprises at least a portion of an immunoglobulin or a variant thereof.

62. The bispecific CAR according to any one of claims 1 to 61, wherein the spacer comprises a hinge region of an immunoglobulin or a variant thereof.

63. The bispecific CAR of claim 62, wherein the immunoglobulin hinge region is an IgG4 hinge region or a variant thereof, optionally a human IgG4 hinge region or a variant thereof.

64. The bispecific CAR of any one of claims 1 to 63, wherein the spacer is less than about 15 amino acids in length.

65. The bispecific CAR according to any one of claims 1 to 64, wherein the spacer is 12 to 15 amino acids in length.

66. The bispecific CAR according to any one of claims 1 to 65, wherein the spacer comprises the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

25.

67. The bispecific CAR according to any one of claims 1 to 64, wherein the spacer is 200 to 250 amino acids in length, optionally 220 to 240 amino acids in length.

68. The bispecific CAR according to any one of claims 1 to 64 and 67, wherein the spacer comprises a hinge region of an immunoglobulin, a CH2 region of an immunoglobulin or a chimeric CH2 region of two different immunoglobulins, and a CH3 region of an immunoglobulin.

69. The bispecific CAR according to any one of claims 1 to 64, 67, and 68, wherein the spacer comprises an IgG4 hinge region or a variant thereof, a chimeric CH2 region comprising a portion of an IgG4 CH2 and a portion of an IgG2 CH2 (IgG2 / 4 CH2 region), and an IgG4 CH3 region.

70. The bispecific CAR according to any one of claims 1 to 64 and 67 to 69, wherein the spacer comprises an amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

27.

71. The bispecific CAR according to any one of claims 1 to 70, wherein the transmembrane domain is or comprises a transmembrane domain of CD4, CD28, or CD8, optionally human CD4, human CD28, or human CD8.

72. The bispecific CAR according to any one of claims 1 to 71, wherein the transmembrane domain is or comprises the transmembrane domain of human CD28.

73. The bispecific CAR according to any one of claims 1 to 72, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

28.

74. The bispecific CAR according to any one of claims 1 to 73, wherein the intracellular signaling domain is a domain derived from a T cell receptor (TCR) component or comprises an immunoreceptor tyrosine-based activation motif (ITAM).

75. The bispecific CAR according to any one of claims 1 to 74, wherein the intracellular signaling domain comprises a cytoplasmic signaling domain of a CD3 zeta chain, optionally a human CD3 zeta chain.

76. The bispecific CAR according to any one of claims 1 to 75, wherein the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

30.

77. The bispecific CAR according to any one of claims 1 to 76, wherein the intracellular signaling domain further comprises a costimulatory signaling region.

78. The bispecific CAR of claim 77, wherein the costimulatory signaling region is located between the transmembrane region and the intracellular signaling domain.

79. The bispecific CAR of claim 77 or 78, wherein the costimulatory signaling region comprises an intracellular signaling domain of a T cell costimulatory molecule or a signaling portion thereof.

80. The bispecific CAR according to any one of claims 77 to 79, wherein the costimulatory signaling region comprises the intracellular signaling domain or a signaling portion thereof of CD28, 4-1BB, or ICOS, optionally human CD28, human 4-1BB, or human ICOS.

81. The bispecific CAR according to any one of claims 77 to 80, wherein the costimulatory signaling region comprises an intracellular signaling domain or a signaling portion thereof of 4-1BB, optionally human 4-1BB.

82. The bispecific CAR according to any one of claims 68 to 72, wherein the costimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:

29.

83. The bispecific CAR according to any one of claims 1 to 82, comprising an amino acid sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 98% sequence identity to any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:

44.

84. The bispecific CAR according to any one of claims 1 to 83, comprising the amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:

44.

85. The bispecific CAR of claim 84, comprising the amino acid sequence set forth in SEQ ID NO:

37.

86. (a) an extracellular domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and a GPRC5D-binding domain that binds to GPRC5D, and a VH region and a VL region and a BCMA-binding domain that binds to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus: (i) a VH region of a GPRC5D binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; (ii) a linker shown in SEQ ID NO: 21; (iii) a VL region of a BCMA-binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; (iv) a linker shown in SEQ ID NO: 17; (v) a VH region of a BCMA-binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; (vi) a linker as set forth in SEQ ID NO: 21, and (vii) comprising a VL region of a GPRC5D-binding domain, including CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively; (b) a spacer comprising the amino acid sequence set forth in SEQ ID NO: 27; (c) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 28; and (d) an intracellular signaling domain comprising the amino acid sequences set forth in SEQ ID NOs: 29 and 30; A bispecific chimeric antigen receptor (CAR), comprising:

87. The bispecific CAR of claim 86, wherein the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:

83.

88. The bispecific CAR according to claim 86 or 87, comprising the amino acid sequence shown in SEQ ID NO:

37.

89. The bispecific CAR according to any one of claims 76 to 88, encoded by the nucleotide sequence shown in SEQ ID NO:

119.

90. (a) an extracellular domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region and a GPRC5D-binding domain that binds to GPRC5D, and a VH region and a VL region and a BCMA-binding domain that binds to BCMA, wherein the extracellular domain comprises, in order from amino to carboxy terminus: (i) a VL region of a BCMA-binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively; (ii) a linker shown in SEQ ID NO: 21; (vii) a VL region of a GPRC5D binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; (iv) a linker shown in SEQ ID NO: 17; (i) a VH region of a GPRC5D binding domain comprising CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; (vi) a linker as set forth in SEQ ID NO: 21, and (v) comprising a VH region of a BCMA-binding domain, including CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; (b) a spacer comprising the amino acid sequence set forth in SEQ ID NO: 27; (c) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 28; and (d) an intracellular signaling domain comprising the amino acid sequences set forth in SEQ ID NOs: 29 and 30; A bispecific chimeric antigen receptor (CAR), comprising:

91. The bispecific CAR of claim 90, wherein the extracellular binding domain comprises the amino acid sequence set forth in SEQ ID NO:

86.

92. The bispecific CAR according to claim 90 or 91, comprising the amino acid sequence shown in SEQ ID NO:

40.

93. The bispecific CAR according to any one of claims 90 to 92, encoded by the polynucleotide sequence shown in SEQ ID NO:

120.

94. A polynucleotide encoding the CAR according to any one of claims 1 to 88 and 90.

95. 95. The polynucleotide of claim 94, comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 105 to 120.

96. A polynucleotide comprising the nucleotide sequence set forth in any one of SEQ ID NOs: 105 to 120.

97. 97. The polynucleotide of any one of claims 94 to 96, which is optimized by splice site removal.

98. 98. The polynucleotide of any one of claims 94 to 97, which is codon-optimized for expression in a human cell.

99. 99. The polynucleotide of any one of claims 94 to 98, comprising the nucleotide sequence set forth in SEQ ID NO: 119 or SEQ ID NO:

120.

100. 100. The polynucleotide of any one of claims 94 to 99, comprising the nucleotide sequence set forth in SEQ ID NO:

119.

101. 101. The polynucleotide of any one of claims 94 to 100, comprising the nucleotide sequence set forth in SEQ ID NO:

120.

102. A vector comprising the polynucleotide of any one of claims 94 to 101.

103. The vector of claim 102, which is a viral vector.

104. 104. The vector of claim 102 or 103, which is a retroviral vector.

105. The vector of any one of claims 102 to 104, which is a lentiviral vector or an adeno-associated viral (AAV) vector.

106. A cell comprising the CAR according to any one of claims 1 to 93.

107. A cell comprising the polynucleotide of any one of claims 90 to 101 or the vector of any one of claims 102 to 105.

108. 108. The cell of claim 106 or 107, which is an immune cell.

109. The cell of any one of claims 106 to 108, which is a lymphocyte.

110. The cell of any one of claims 106 to 109, which is a NK cell or a T cell.

111. The cell of any one of claims 106 to 110, which is a T cell.

112. The cell of claim 111, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

113. 113. The cell of claim 111 or 112, wherein the T cell is a primary T cell.

114. 108. The cell of claim 106 or 107, which is a stem cell.

115. The cell of claim 114, wherein the stem cell is a multipotent or pluripotent stem cell.

116. 116. The cell of claim 114 or 115, wherein the stem cell is an induced pluripotent stem cell (iPSC).

117. The cell according to any one of claims 106 to 112, which is a cell differentiated from an induced pluripotent stem cell.

118. 118. The cell of any one of claims 106 to 117, which is an allogeneic cell.

119. The cell of any one of claims 106 to 118, which has been engineered to be hypoimmune.

120. The cell of any one of claims 98 to 119, which exhibits cytotoxic activity against GPRC5D+ cells, BCMA+ cells, or GPRC5D+ / BCMA+ cells.

121. A composition comprising a plurality of cells according to any one of claims 106 to 120.

122. 122. The composition of claim 121, further comprising a pharmaceutically acceptable excipient.

123. 121. A pharmaceutical composition comprising a plurality of cells according to any one of claims 106 to 120, and a pharmaceutically acceptable excipient.

124. 124. The composition of any one of claims 121 to 123, comprising CD4+ T cells and CD8+ T cells.

125. 125. The composition of claim 124, comprising CD4+ T cells and CD8+ T cells in a ratio of about 1:3 to about 3:1, optionally in a ratio of about 1:2 to about 2:1, and further optionally in a ratio of about 1:

1.

126. 126. The composition of claim 124 or 125, comprising CD4+ T cells and CD8+ T cells in a ratio of about 1:3 to about 3:

1.

127. 127. The composition of any one of claims 124 to 126, comprising CD4+ T cells and CD8+ T cells in a ratio of about 1:

1.

128. 128. The composition of any one of claims 121-127, wherein greater than about 90%, greater than about 95%, or greater than about 99% of the cells in the composition are CD3+ T cells.

129. 129. The composition of any one of claims 121-128, wherein at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of cells in the composition express the CAR.

130. 130. The composition of any one of claims 121-129, wherein, of the plurality of cells in the composition that express the CAR, less than about 10%, about 9%, about 8%, about 7%, about 5%, about 4%, about 3%, about 2%, or about 1% of the cells exhibit sustained signaling.

131. Including the end points, approximately 1.0 x 10 ７ 1.2 × 10 from CAR-expressing T cells ９ CAR-expressing T cells, approximately 1.0 x 10 ７ 6.5 × 10 from CAR-expressing T cells ８ CAR-expressing T cells, approximately 1.5 x 10 ７ 6.5 × 10 from CAR-expressing T cells ８ CAR-expressing T cells, approximately 1.5 x 10 ７ 6.0 × 10 from CAR-expressing T cells ８ CAR-expressing T cells, approximately 2.5 x 10 ７ 6.0 × 10 from CAR-expressing T cells ８ CAR-expressing T cells, approximately 5.0 x 10 ７ 6.0 × 10 from CAR-expressing T cells ８ CAR-expressing T cells, approximately 1.25 x 10 ７ 1.2 × 10 from CAR-expressing T cells ９ CAR-expressing T cells, approximately 1.5 x 10 ７ 1.2 × 10 from CAR-expressing T cells ９ CAR-expressing T cells, approximately 5.0 x 10 ７ 4.5 × 10 from CAR-expressing T cells ８ CAR-expressing T cells, or about 1.5 x 10 ８ 3.0 × 10 from CAR-expressing T cells ８ The composition of any one of claims 121 to 130, comprising a CAR-expressing T cell.

132. Approximately 1.5×10 ７ , about 2.5×10 ７ , about 5.0×10 ７ , about 7.5×10 ７ , about 1.0×10 ８ , about 1.25×10 ８ , about 1.5×10 ８ , about 1.75×10 ８ , about 2×10 ８ , about 2.25×10 ８ , about 2.5×10 ８ , about 3.0×10 ８ , about 3.5×10 ８ , about 4×10 ８ , about 4.5×10 ８ , about 6.0×10 ８ , about 8.0×10 ８ , or about 1.2 × 10 ９ The composition of any one of claims 121 to 131, comprising CAR-expressing T cells.

133. 133. A method of treating a disease or condition comprising administering to a subject a cell according to any one of claims 106 to 120 or a composition according to any one of claims 121 to 132.

134. The cells were approximately 1 x 10 ７ 1 x 10 from CAR-expressing T cells ９ 134. The method of claim 133, wherein the CAR-expressing T cells are administered to the subject in a dose of up to 100 mg / kg of the CAR-expressing T cells.

135. The cells were approximately 2.5 x 10 ７ Approximately 4.5 × 10 from CAR-expressing T cells ８ 134. The method of claim 133, wherein the CAR-expressing T cells are administered to the subject in a dose of up to 100 mg / kg of the CAR-expressing T cells.

136. The cells were approximately 2.5 x 10 ７ 136. The method of any one of claims 133-135, wherein the CAR-expressing T cells are administered to the subject in a dose of

137. The cells were approximately 7.5 x 10 ７ 136. The method of any one of claims 133-135, wherein the CAR-expressing T cells are administered to the subject in a dose of

138. The cells were approximately 1.5 x 10 ８ 136. The method of any one of claims 133-135, wherein the CAR-expressing T cells are administered to the subject in a dose of

139. The cells were approximately 3.0 × 10 ８ 136. The method of any one of claims 133-135, wherein the CAR-expressing T cells are administered to the subject in a dose of

140. The cells were approximately 4.5 x 10 ８ 136. The method of any one of claims 133-135, wherein the CAR-expressing T cells are administered to the subject in a dose of

141. 141. The method of any one of claims 133-140, further comprising administering to the subject a lymphocyte depleting therapy prior to administration of the dose of CAR-expressing T cells.

142. 142. The method of any one of claims 133-141, wherein lymphocyte depletion therapy is completed within about 7 days prior to initiation of administration of doses of CAR-expressing T cells.

143. 143. The method of any one of claims 133-142, wherein administration of the lymphocyte depleting therapy is completed within about 2 to 7 days prior to initiating administration of the dose of engineered T cells.

144. 144. The method of any one of claims 133 to 143, wherein the lymphocyte depleting treatment comprises the administration of fludarabine and / or cyclophosphamide.

145. 145. The method of any one of claims 133 to 144, wherein the lymphocyte depleting therapy comprises administration of fludarabine and cyclophosphamide.

146. Lymphocyte depletion therapy is administered at approximately 200-400 mg / m daily, including endpoints. ２ 146. The method of any one of claims 133 to 145, comprising administering cyclophosphamide in a

147. Lymphocyte depletion therapy is administered at approximately 300 mg / m daily. ２ 147. The method of any one of claims 133 to 146, comprising administering cyclophosphamide in a

148. Lymphocyte depletion therapy is administered at approximately 20-40 mg / m daily, including endpoints. ２ 146. The method of any one of claims 133 to 145, comprising administering fludarabine in a

149. Lymphocyte depletion therapy is administered at approximately 30 mg / m daily. ２ 149. The method of any one of claims 133 to 146 and 148, comprising administering fludarabine in a dose of 100 mg / kg.

150. 150. The method of any one of claims 133 to 149, wherein the lymphocyte depleting treatment comprises administration of fludarabine and cyclophosphamide for 2 to 4 days.

151. 151. The method of any one of claims 133 to 150, wherein the lymphocyte depleting treatment comprises administration of fludarabine and cyclophosphamide for three days.

152. 144. The method of any one of claims 133 to 143, wherein the lymphocyte depleting treatment comprises administration of bendamustine.

153. Lymphocyte depletion therapy is administered at approximately 50-130 mg / m daily, including endpoints. ２ 153. The method of any one of claims 133-143 and 152, comprising administering bendamustine in a

154. Lymphocyte depletion therapy is approximately 90 mg / m daily. ２ 154. The method of any one of claims 133-143, 152, and 153, comprising administering bendamustine in a

155. 155. The method of any one of claims 133-143 and 152-154, wherein the lymphocyte depleting treatment comprises administration of bendamustine for 1 to 3 days.

156. 156. The method of any one of claims 133-143 and 152-155, wherein the lymphocyte depleting treatment comprises administration of bendamustine for two days.

157. 157. The method of any one of claims 133 to 156, wherein the disease or condition is cancer, optionally a plasma cell malignancy.

158. The method of any one of claims 133 to 157, wherein the disease or condition is a BCMA-expressing cancer and / or a GPRC5D-expressing cancer.

159. 159. The method of any one of claims 133 to 158, wherein the disease or condition is multiple myeloma.

160. 160. The method of any one of claims 133 to 159, wherein the disease or condition is relapsed / refractory multiple myeloma (RRMM).

161. 161. The method of any one of claims 133 to 160, wherein the subject has undergone one or more prior therapies.

162. 162. The method of any one of claims 133-161, wherein the subject has received at least one but no more than three prior therapies.

163. 163. The method of claim 161 or 162, wherein the prior treatment is a proteasome inhibitor, an immunomodulator, an anti-CD38 antibody, prior treatment included in autologous hematopoietic stem cell transplantation (HSCT), or any combination thereof.

164. 133. Use of a cell according to any one of claims 106 to 120 or a composition according to any one of claims 121 to 132 in the manufacture of a medicament for treating a disease or condition in a subject.

165. Use of a cell according to any one of claims 106 to 120 or a composition according to any one of claims 121 to 132 for treating a disease or condition in a subject.

166. 166. Use according to claim 164 or 165, wherein the disease or condition is cancer, optionally a plasma cell malignancy.

167. The use according to any one of claims 164 to 166, wherein the disease or condition is a BCMA-expressing cancer and / or a GPRC5D-expressing cancer.

168. 168. The use of any one of claims 164 to 167, wherein the disease or condition is multiple myeloma.

169. 169. The use of any one of claims 164 to 168, wherein the disease or condition is relapsed / refractory multiple myeloma (RRMM).

170. 170. The use of any one of claims 164 to 169, wherein the subject has undergone one or more prior treatments.

171. 170. The use of any one of claims 164 to 169, wherein the subject has received at least one but not more than three prior therapies.

172. 172. The use of claim 170 or claim 171, wherein the prior treatment is a proteasome inhibitor, an immunomodulator, an anti-CD38 antibody, prior treatment included in autologous hematopoietic stem cell transplantation (HSCT), or any combination thereof.

173. A cell according to any one of claims 106 to 120 or a composition according to any one of claims 121 to 132 for treating a disease or condition in a subject.

174. 174. The cell or composition of claim 173, wherein the disease or condition is cancer, optionally a plasma cell malignancy.

175. The cell or composition of claim 173 or 174, wherein the disease or condition is a BCMA-expressing cancer and / or a GPRC5D-expressing cancer.

176. 176. The cell or composition of any one of claims 173 to 175, wherein the disease or condition is multiple myeloma.

177. 177. The cell or composition of any one of claims 173 to 176, wherein the disease or condition is relapsed / refractory multiple myeloid leukemia (RRMM).

178. 178. The cell or composition of any one of claims 173 to 177, wherein the subject has undergone one or more prior therapies.

179. 179. The cell or composition of any one of claims 173-178, wherein the subject has received at least one but not more than three prior therapies.

180. The cell or composition of claim 178 or 179, wherein the prior treatment is a proteasome inhibitor, an immunomodulator, an anti-CD38 antibody, prior treatment included in autologous hematopoietic stem cell transplantation (HSCT), or any combination thereof.

181. A kit comprising the CAR according to any one of claims 1 to 93, the polynucleotide according to any one of claims 94 to 101, the vector according to any one of claims 102 to 105, the cell according to any one of claims 106 to 120, or the composition according to any one of claims 121 to 132, and an instruction manual for use, optionally wherein the instruction manual is for administering the CAR, the cell, or the composition.

182. The kit of claim 181, wherein the instructions specify administering the CAR, cell, or composition to a subject having a disease or condition.

183. An article of manufacture comprising a CAR according to any one of claims 1 to 93, a polynucleotide according to any one of claims 94 to 101, a vector according to any one of claims 102 to 105, a cell according to any one of claims 106 to 120, a composition according to any one of claims 121 to 132, or a kit according to claim 181 or 182.

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