GPRC5D and BCMA specific chimeric antigen receptor

Abstract

A chimeric antigen receptor (CAR) is provided that contains an extracellular antigen binding domain that binds to a G protein coupled receptor class C Group 5 D member (GPRC5D) and a B cell maturation antigen (BCMA). The disclosure also relates to genetically engineered cells expressing such CARs and their use in adoptive cell therapy.

Description

Cross - reference to related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 395,702, filed on Aug. 5, 2022, entitled "GPRC5D and BCMA Specific Chimeric Antigen Receptors", the content of which is incorporated herein by reference in its entirety. Technical Field

[0002] In some aspects, the present disclosure relates to chimeric antigen receptors (CARs) that contain extracellular antigen-binding domains that bind to G protein-coupled receptor class C group 5 member D (GPRC5D) and B-cell maturation antigen (BCMA). The present disclosure also relates to genetically engineered cells that express such CARs and their use in adoptive cell therapy. Incorporation by reference of the Sequence Listing

[0003] This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named 735042026340SeqList.xml, created on Aug. 4, 2023, and having a size of 224,174 bytes. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety. Background Art

[0004] G protein-coupled receptor class C group 5 member D (GPRC5D) is a G protein-coupled receptor that is minimally expressed in bone marrow samples from patients with other hematological malignancies but is highly expressed in bone marrow samples from patients with multiple myeloma (MM). 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 is still a need for improved CARs that bind both GPRC5D and BCMA, as well as engineered cells that express both GPRC5D and BCMA, for use in adoptive cell therapy. This application provides embodiments that meet such needs. Summary of the Invention

[0005] This application provides bispecific chimeric antigen receptors (CARs) that contain an extracellular domain that contains a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA.

[0006] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl terminus: (i) 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; or (ii) 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; (b) a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.

[0007] In some embodiments, the extracellular domain sequentially comprises (i) from the amino terminus to the carboxyl terminus. In some embodiments, the extracellular domain sequentially comprises from the amino terminus to the carboxyl 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.

[0008] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl 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 sequentially comprises from the amino terminus to the carboxyl 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.

[0010] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain that comprises a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain comprises, in order from the amino terminus to the carboxyl 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, in order from the amino terminus to the carboxyl 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.

[0012] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain that comprises a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain comprises, in order from the amino terminus to the carboxyl 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, in order from the amino terminus to the carboxyl 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.

[0014] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl 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 sequentially comprises (ii) from the amino terminus to the carboxyl terminus. In some embodiments, the extracellular domain sequentially comprises from the amino terminus to the carboxyl 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.

[0016] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl 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 sequentially comprises from the amino terminus to the carboxyl 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.

[0018] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain that contains a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain contains a heavy chain variable (VH) region and a light chain variable (VL) region, and the BCMA-binding domain contains a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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 sequentially comprises, from the amino terminus to the carboxyl 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.

[0020] The present application also provides a bispecific chimeric antigen receptor, which comprises: (a) an extracellular domain that contains a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain contains a heavy chain variable (VH) region and a light chain variable (VL) region, and the BCMA-binding domain contains a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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 sequentially comprises, from the amino terminus to the carboxyl 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.

[0022] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region, and the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain comprises, in order from the amino terminus to the carboxyl 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 region or the VL region of the GPRC5D-binding domain; and (b) the VH region or the VL region of the BCMA-binding domain are linked by a linker.

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

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

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

[0027] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl terminus: (i) the VH region of the GPRC5D-binding domain; (ii) the linker shown in SEQ ID NO: 21; (iii) the VL region of the BCMA-binding domain; (iv) the linker shown in SEQ ID NO: 17; (v) the VH region of the BCMA-binding domain; (vi) the linker shown 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] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising (i) a GPRC5D-binding domain that binds to GPRC5D, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) a BCMA-binding domain that binds to BCMA, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl 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) an intracellular signaling domain.

[0029] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising (i) a GPRC5D binding domain that binds to GPRC5D, the GPRC5D binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) a BCMA binding domain that binds to BCMA, the BCMA binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl terminus: the VL region of the GPRC5D binding domain; the VH region of the GPRC5D binding domain; one 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 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 length of the linker is 4-12 amino acids. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:24. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:19. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:21. In some embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:24. In some embodiments, the VH region and the VL region of the BCMA binding domain are linked by a linker comprising the amino acid sequence shown in SEQ ID NO:17.

[0031] The present invention also provides a bispecific chimeric antigen receptor (CAR), comprising: (a) an extracellular domain, the extracellular domain comprising (i) a GPRC5D-binding domain that binds to GPRC5D, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) a BCMA-binding domain that binds to BCMA, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl terminus: the VH region of the GPRC5D-binding domain; the VL region of the GPRC5D-binding domain; one 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 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 shown in SEQ ID NO:19 or SEQ ID NO:21.

[0032] In some embodiments, the VH region of the GPRC5D-binding domain comprises CDR-1, CDR-2, and CDR-3 containing the amino acid sequences shown 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 containing the amino acid sequences shown 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 containing the amino acid sequences shown 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 containing the amino acid sequences shown 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 with the amino acid sequence shown 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 with the amino acid sequence shown 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 with 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 with the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the VH region of the GPRC5D-binding domain comprises the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the VL region of the GPRC5D-binding domain comprises the amino acid sequence shown in SEQ ID NO:8. In some embodiments, 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.

[0033] In some embodiments, the VH region of the BCMA-binding domain comprises CDR-1, CDR-2, and CDR-3 containing the amino acid sequences shown 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 containing the amino acid sequences shown 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 with the amino acid sequence shown 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 with the amino acid sequence shown 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 with 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 with the amino acid sequence shown in SEQ ID NO:16. In some embodiments, the VH region of the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO:15. In some embodiments, the VL region of the BCMA-binding domain comprises the amino acid sequence shown in SEQ ID NO:16. In some embodiments, 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.

[0034] In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown in any one of SEQ ID NO: 77, 78, 79, and 80. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown in SEQ ID NO: 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 shown in SEQ ID NO: 83. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown in SEQ ID NO: 87. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown in SEQ ID NO: 81. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown in SEQ ID NO: 85. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown in SEQ ID NO: 86. In some embodiments, the extracellular binding domain of the bispecific CAR comprises the amino acid sequence shown 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 the hinge region of an immunoglobulin or a variant thereof. In some embodiments, the hinge region of the immunoglobulin is the IgG4 hinge region. In some embodiments, the hinge region comprises the human IgG4 hinge region or a variant thereof.

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

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

[0038] In some embodiments, the intracellular signaling domain is a domain 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 CD3ζ chain. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of the human CD3ζ chain. In some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:30. In some embodiments, the intracellular signaling domain comprises the amino acid sequence shown in SEQ ID NO:30. In some embodiments, the intracellular signaling region comprises a co-stimulatory signaling region. In some embodiments, the co-stimulatory signaling region is located between the transmembrane region and the intracellular signaling domain. In some embodiments, the co-stimulatory signaling region comprises the intracellular signaling domain or a signaling portion thereof of a T cell co-stimulatory molecule. In some embodiments, the co-stimulatory signaling region comprises the intracellular signaling domain or a signaling portion thereof of CD28, 4-1BB, or ICOS. In some embodiments, the co-stimulatory signaling region comprises the intracellular signaling domain of human CD28, human 4-1BB, or human ICOS. In some embodiments, the co-stimulatory signaling region comprises the intracellular signaling domain or a signaling portion thereof of 4-1BB. In some embodiments, the co-stimulatory signaling region comprises the signal transduction domain of human 4-1BB. In some embodiments, the co-stimulatory signaling region comprises the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least about 90% sequence identity to the amino acid sequence shown in SEQ ID NO:29. In some embodiments, the co-stimulatory signaling region comprises the amino acid sequence shown 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 99% 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] It should be noted that there seems to be a repetition in the original text where it says "at least about 98%" twice. I translated it as "at least about 99%" in the translation to make the range more sequential. If this is not what you intended, please let me know.In some embodiments, the CAR comprises 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. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:31. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:32. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:33. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:34. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:35. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:36. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:37. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:38. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:39. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:40. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:42. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:43. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:44.

[0041] The present application also provides a bispecific chimeric antigen receptor (CAR), which comprises: (a) an extracellular domain, the extracellular domain comprising a GPRC5D binding domain that binds to GPRC5D and a BCMA binding domain that binds to BCMA, the GPRC5D binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the BCMA binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl terminus: (i) the VH region of the GPRC5D binding domain, the VH region comprising CDR-1, CDR-2 and CDR-3 containing the amino acid sequences shown 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) the VL region of the BCMA binding domain, the VL region comprising CDR-1, CDR-2 and CDR-3 containing the amino acid sequences shown 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) the VH region of the BCMA binding domain, the VH region comprising CDR-1, CDR-2 and CDR-3 containing the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11 respectively; (vi) a linker shown in SEQ ID NO:21; and (vii) the VL region of the GPRC5D binding domain, the VL region comprising CDR-1, CDR-2 and CDR-3 containing the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively; (b) a spacer comprising the amino acid sequence shown in SEQ ID NO:27; (c) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:28; and (d) an intracellular signaling domain comprising the amino acid sequences shown in SEQ ID NO:29 and 30.

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

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

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

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

[0046] The present application also provides a vector comprising any polynucleotide 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 virus (AAV) vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0047] The present application also provides a cell comprising any CAR provided herein.

[0048] The present application also provides a cell comprising any polynucleotide provided herein.

[0049] The present application also provides a cell comprising any vector provided herein. In some embodiments, the cell is an immune cell. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is an NK cell or a T cell. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a CD4+ T cell or a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell. In some embodiments, the T cell is a primary T cell. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell has differentiated from an induced pluripotent stem cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is engineered to be hypoimmunogenic.

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

[0051] The present application also provides a composition comprising a plurality of any cells provided herein. In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0052] The present application also provides a pharmaceutical composition, which comprises a plurality of any cells 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, and the ratio is between about 1:3 and about 3:1. In some embodiments, the composition comprises a ratio of CD4+ T cells to CD8+ T cells, and the ratio is between about 1:2 and about 2:1. In some embodiments, the composition comprises a ratio of CD4+ T cells to CD8+ T cells, and the ratio is about 1:1.

[0054] In some embodiments, greater than about 90%, greater than about 95% or greater than about 99% 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 the CAR. In some embodiments, among a plurality of cells expressing the CAR in the composition, 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 tonic signaling.

[0055] In some embodiments, the composition comprises about 1.0x10 7 CAR-expressing T cells to 1.2x10 9 CAR-expressing T cells, between about 1.0x10 7 CAR-expressing T cells to 6.5x10 8 CAR-expressing T cells, between about 1.5x10 7 CAR-expressing T cells to 6.5x10 8 CAR-expressing T cells, between about 1.5x10 7 CAR-expressing T cells to 6.0x10 8 CAR-expressing T cells, between about 2.5x10 7 CAR-expressing T cells to 6.0x10 8 CAR-expressing T cells, between about 5.0x10 7 CAR-expressing T cells to 6.0x10 8 CAR-expressing T cells, between about 1.25x10 7 CAR-expressing T cells to 1.2x10 9 CAR-expressing T cells, between about 1.5x10 7 CAR-expressing T cells to 1.2x10 9Between about 5.0x10 CAR-expressing T cells, about 5.0x10 7 CAR-expressing T cells and about 4.5x10 8 CAR-expressing T cells, or between about 1.5x10 8 CAR-expressing T cells and about 3.0x10 8 CAR-expressing T cells, with each endpoint value included. In some embodiments, the composition comprises about 1.5x10 7 CAR-expressing T cells, about 2.5x10 7 CAR-expressing T cells, about 5.0x10 7 CAR-expressing T cells, about 7.5x10 7 CAR-expressing T cells, about 1.0x10 8 CAR-expressing T cells, about 1.25x10 8 CAR-expressing T cells, about 1.5x10 8 CAR-expressing T cells, about 1.75x10 8 CAR-expressing T cells, about 2x10 8 CAR-expressing T cells, about 2.25x10 8 CAR-expressing T cells, about 2.5x10 8 CAR-expressing T cells, about 3.0x10 8 CAR-expressing T cells, about 3.5x10 8 CAR-expressing T cells, about 4x10 8 CAR-expressing T cells, about 4.5x10 8 CAR-expressing T cells, about 6.0x10 8 CAR-expressing T cells, about 8.0x10 8 , or about 1.2x10 9 CAR-expressing T cells.

[0056] The present application also provides a method of treating a disease or disorder, the method comprising administering to a subject any of the cells provided herein. In some embodiments, the cells are administered to the subject at a dose of from about 1x10 7 CAR-expressing T cells to 1x10 9 CAR-expressing T cells. In some embodiments, the cells are administered to the subject at a dose of from about 2.5x10 7 CAR-expressing T cells to about 4.5x10 8 CAR-expressing T cells. In some embodiments, the cells are administered to the subject at a dose of from about 2.5x10 7 CAR-expressing T cells. In some embodiments, the cells are administered to the subject at a dose of from about 7.5x10 7 CAR-expressing T cells. In some embodiments, the cells are administered to the subject at a dose of from about 1.5x10 8A dose of CAR-expressing T cells is administered to the subject. In some embodiments, the cells are administered at a dose of or about 3.0x10 8 A dose of CAR-expressing T cells is administered to the subject. In some embodiments, the cells are administered at a dose of or about 4.5x10 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 lymphodepletion therapy to the subject prior to administration of the dose of CAR-expressing T cells. In some embodiments, the lymphodepletion therapy is completed within about 7 days prior to the initiation of administration of the dose of CAR-expressing T cells. In some embodiments, the administration of the lymphodepletion therapy is completed within about 2 to 7 days prior to the administration of the dose of engineered T cells. In some embodiments, the lymphodepletion therapy comprises the administration of fludarabine and / or cyclophosphamide. In some embodiments, the lymphodepletion therapy comprises the administration of fludarabine and cyclophosphamide. In some embodiments, the lymphodepletion therapy comprises administering daily a cyclophosphamide of or about 200-400 mg / m 2 with end values included. In some embodiments, the lymphodepletion therapy comprises administering daily a cyclophosphamide of or about 300 mg / m 2 of cyclophosphamide. In some embodiments, the lymphodepletion therapy comprises administering daily a fludarabine of or about 20-40 mg / m 2 with end values included. In some embodiments, the lymphodepletion therapy comprises administering daily a fludarabine of or about 30 mg / m 2 of fludarabine. In some embodiments, the lymphodepletion therapy comprises administering fludarabine and cyclophosphamide for 2-4 days. In some embodiments, the lymphodepletion therapy comprises administering fludarabine and cyclophosphamide for 3 days.

[0058] In some embodiments, the lymphodepletion therapy comprises the administration of bendamustine. In some embodiments, the lymphodepletion therapy comprises administering daily a bendamustine of or about 50-130 mg / m 2 with end values included. In some embodiments, the lymphodepletion therapy comprises administering daily a bendamustine of or about 90 mg / m 2 of bendamustine. In some embodiments, the lymphodepletion therapy comprises administering bendamustine for 1-3 days. In some embodiments, the lymphodepletion therapy comprises administering bendamustine for 2 days.

[0059] The present application also provides the use of any cell provided herein for the preparation of a medicament for treating a disease or disorder of a subject. The present application also provides the use of any cell provided herein for treating a disease or disorder of a subject. The present application also provides any cell provided herein for treating a disease or disorder of a subject.

[0060] The present application also provides a method for treating a disease or disorder, the method comprising administering to a subject any composition provided herein. The present application also provides the use of any composition provided herein for the preparation of a medicament, the pharmaceutical composition being for treating a disease or disorder of a subject. The present application also provides the use of any composition provided herein for treating a disease or disorder of a subject. The present application also provides any composition provided herein for treating a disease or disorder of a subject.

[0061] In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is a plasma cell malignancy. In some embodiments, the disease or disorder is a cancer expressing BCMA and / or a cancer expressing GPRC5D. In some embodiments, the disease or disorder is a cancer expressing BCMA. In some embodiments, the disease or disorder is a cancer expressing GPRC5D. In some embodiments, the disease or disorder is a cancer expressing BCMA and a cancer expressing GPRC5D. In some embodiments, the disease or disorder is multiple myeloma. In some embodiments, the disease or disorder 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 1 but no more than 3 prior therapies. In some embodiments, the prior therapy is a proteasome inhibitor, an immunomodulatory agent, an anti-CD38 antibody, a prior therapy comprising autologous hematopoietic stem cell transplantation (HSCT), or any combination of the foregoing. In some embodiments, the cell or composition can be used for the preparation of a medicament for the treatment of a disease or disorder of a subject. In some embodiments, the cell or composition can be used to treat a disease or disorder of a subject. In some embodiments, the disease or disorder is cancer, optionally a plasma cell malignancy. In some embodiments, the disease or disorder is a cancer expressing BCMA and / or a cancer expressing GPRC5D. In some embodiments, the disease or disorder is multiple myeloma. In some embodiments, the disease or disorder 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 1 but no more than 3 prior therapies. In some embodiments, the prior therapy is a proteasome inhibitor, an immunomodulator, an anti-CD38 antibody, a prior therapy comprising autologous hematopoietic stem cell transplantation (HSCT), or any combination of the foregoing. In some embodiments, the cell or composition can be used to treat a disease or disorder in a subject. In some embodiments, the disease or disorder is cancer, optionally a plasma cell malignancy. In some embodiments, the disease or disorder is a cancer expressing BCMA and / or a cancer expressing GPRC5D. In some embodiments, the disease or disorder is multiple myeloma. In some embodiments, the disease or disorder 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 1 but no more than 3 prior therapies. In some embodiments, the prior therapy is a proteasome inhibitor, an immunomodulator, an anti-CD38 antibody, a prior therapy comprising autologous hematopoietic stem cell transplantation (HSCT), or any combination of the foregoing.

[0064] The present application also provides a kit, the kit comprising any CAR, polynucleotide, vector, cell, or composition provided herein and instructions for use. In some embodiments, the instructions are for administering the CAR, the cell, or the composition. In some embodiments, the instructions specifically state to administer the CAR, the cell, or the composition to a subject having a disease or disorder.

[0065] The present application also provides an article of manufacture, the article of manufacture comprising any CAR, polynucleotide, vector, cell, composition, or kit provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1A and 1B shows the expression of human GPRC5D and human BCMA on various tumor cell lines as determined by flow cytometry.

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

[0068] Figure 2BShows the structure of an exemplary generated bispecific, circular tandem CAR targeting GPRC5D and BCMA, which has a GPRC5D binding domain (left panel) or a BCMA binding domain (right panel) proximal to the cell membrane.

[0069] Figure 3A and 3B Show the antigen-independent (tonic) signaling ( Figure 3A ) and antigen-dependent signaling ( Figure 3B ) of Nurkat reporter cells expressing an exemplary generated bispecific tandem CAR alone or after co-culture with target cells expressing GPRC5D and BCMA.

[0070] Figure 4A and 4B Show the antigen-dependent activation of Nurkat reporter cells expressing an exemplary generated bispecific tandem CAR via a single antigen after co-culture with MM.1S or OPM-2 cells in which GPRC5D or BCMA has been knocked out, respectively.

[0071] Figure 5 Shows the percentage of the cell surface that is positive for each CAR expression as determined by flow cytometry (the names of the first 14 tandem CAR constructs are represented by numbers).

[0072] Figure 6A Shows the ability of T cells expressing the indicated tandem CAR constructs to lyse target cells (from left to right: MM.1S, MM.1S BCMA KO, and MM.1S GPRC5D KO) after 21 days of co-culture. *CAR T cells against MM.1S BCMA KO cells did not survive until day 21. ^CAR T cells against MM.1S GPRC5D KO cells did not survive until day 21.

[0073] Figure 6B and 6C Show the proliferation of CAR T cells expressing linear tandem CAR constructs (dots), circular tandem CAR constructs (dots), single-target (GPRC5D or BCMA) CAR constructs (squares and diamonds, respectively), or bicistronic CAR constructs (triangles) after 7, 14, and 21 days of co-culture with MM.1S cells in which BCMA ( Figure 6B ) or GPRC5D ( Figure 6C ) has been knocked out, respectively.

[0074] Figure 7A and 7B Show, respectively, with high dose (2x10 6 )( Figure 7A ) or low dose (0.5x106 )( Figure 7B Individual plots of tumor burden up to day 49 in the MM.1S murine model of multiple myeloma after T cell therapy with a CAR expressing the indicated Figure 7B

[0075] Figure 8A and 8B show the tumor control index (TCI) up to day 49 in the MM.1S multiple myeloma murine model after T cell therapy with a CAR expressing the indicated 8B at high dose (2x10 6 ) or low dose (0.5x10 6 ).

[0076] Figure 9A and 9B show individual plots of tumor burden up to day 49 in the RPMI-8226 murine model of multiple myeloma after T cell therapy with a CAR expressing the indicated 9B at high dose (2x10 6 )( Figure 9A ) or low dose (0.5x10 6 )( Figure 9B ).

[0077] Figure 10A and 10B show the tumor control index (TCI) up to day 49 in the RPMI-8226 multiple myeloma murine model after T cell therapy with a CAR expressing the indicated 10B at high dose (2x10 6 ) or low dose (0.5x10 6 ).

[0078] Figure 11A shows individual plots of tumor burden up to day 28 in a murine model of multiple myeloma antigenic heterogeneity after treatment with 4x10 6 CAR-expressing T cells (solid line) or mock-treated T cells (dashed line).

[0079] Figure 11B and 11C show the tumor control index (TCI) and tumor burden (BLI) up to day 28 by bioluminescence imaging (BLI) in a murine model of multiple myeloma antigenic heterogeneity after treatment with 4x10 6 CAR-expressing T cells.

[0080] Figure 12A and 12B show the expression of both anti-GPRC5D scFv (y-axis) and anti-BCMA scFv (x-axis) in T cells transduced with tandem CAR5, anti-BCMACAR, or anti-GPRC5D CAR or mock from 3 human donors.

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

[0082] Figure 13C and 13D show the secretion of IFNγ ( Figure 13C , upper panel), IL-2 ( Figure 13C , lower panel), and TNFα ( Figure 13D ) of T cells transduced with bispecific tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR or mimics after co-culture with various cell lines. The graphs show the mean concentration of pro-inflammatory cytokines, and the data points represent the cytokine levels of individual donors.

[0083] Figure 14A show the CD25 expression of T cells transduced with bispecific tandem CAR5, anti-BCMA CAR, or anti-GPRC5D CAR or mimics after co-culture with various cell lines. The data points represent the values of CAR T cells from individual donors.

[0084] Figure 14B and 14C show the secretion of IFNγ, IL-2, and TNFα (left panel, middle panel, and right panel, respectively) of 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. The graphs show the mean concentration of pro-inflammatory cytokines, and the data points represent the values from individual donors.

[0085] Figure 15 show the cytotoxic activity of CAR5 T cells, anti-BCMA CAR T cells, and anti-GPRC5D CAR T cells against tumor cell lines expressing various levels of BCMA and GPRC5D. The data are presented as the mean and standard deviation of 3 donors.

[0086] Figure 16A show the number of CAR+ human CD3+ T cells per microliter of peripheral blood in MM.1S xenograft mice treated with 5x10 5 (low dose; left panel) or 2x10 6 (high dose; right panel) CAR T cells.

[0087] Figure 16B (upper panel) shows the number of CAR+ human CD3+ T cells per microliter of peripheral blood in MM.1S xenograft mice treated with 5x105 (Low dose; left panel) or 2 x 10 6 (High dose; right panel) of bispecific tandem CAR5 T cells, anti-BCMA CAR T cells, or anti-GPRC5D CAR T cells, or mock-transduced T cells in MM.1S xenograft mice. Figure 16B (Lower panel) shows the individual tumor volumes in MM.1S xenograft mice treated with 5 x 10 5 (Low dose; left panel) or 2 x 10 6 (High dose; right panel) of anti-GPRC5D CAR T cells or mock-transduced T cells.

[0088] Figure 16C (Upper panel) shows the individual tumor volumes in MM.1S xenograft mice treated with 5 x 10 5 (Low dose; left panel) or 2 x 10 6 (High dose; right panel) of anti-BCMA CAR T cells or mock-transduced T cells. Figure 16C (Lower panel) shows the individual tumor volumes in MM.1S xenograft mice treated with 5 x 10 5 (Low dose; left panel) or 2 x 10 6 (High dose; right panel) of bispecific tandem CAR5 T cells or mock-transduced T cells.

[0089] Figure 16D Shows the tumor control index in MM.1S xenograft mice treated with 5 x 10 5 (Low dose) or 2 x 10 6 (High dose) of tandem CAR5 T cells, anti-BCMA CAR T cells, or anti-GPRC5D CAR T cells, or mock-transduced T cells.

[0090] Figure 16E Shows the survival probability in MM.1S xenograft mice treated with 5 x 10 5 (Low dose) or 2 x 10 6 (High dose) of tandem CAR5 T cells, anti-BCMA CAR T cells, or anti-GPRC5D CAR T cells, or mock-transduced T cells.

[0091] Figure 17A (Upper panel) shows the mean tumor burden in each group of OPM-2 xenograft mice treated with 5 x 10 5 (Low dose) or 2 x 10 6 (High dose) of tandem CAR5 T cells, anti-BCMA CAR T cells, or anti-GPRC5D CAR T cells, or mock-transduced T cells. Figure 17A(The figure below) shows the individual tumor burdens of OPM-2 xenograft mice treated with 5x10 5 (low dose) or 2x10 6 (high dose) anti-GPRC5D CAR T cells or mock-transduced T cells.

[0092] Figure 17B (The figure above) shows the individual tumor burdens of OPM-2 xenograft mice treated with 5x10 5 (low dose) or 2x10 6 (high dose) anti-BCMA CAR T cells or mock-transduced T cells. Figure 17B (The figure below) shows the individual tumor burdens of OPM-2 xenograft mice treated with 5x10 5 (low dose) or 2x10 6 (high dose) tandem CAR5 T cells or mock-transduced T cells.

[0093] Figure 17C Shows the tumor control index of OPM-2 xenograft mice treated with 5x10 5 (low dose) or 2x10 6 (high dose) tandem CAR5 T cells, anti-BCMA CAR T cells or anti-GPRC5D CAR T cells or mock-transduced T cells.

[0094] Figure 17D Shows the survival probability of OPM-2 xenograft mice treated with 5x10 5 (low dose) or 2x10 6 (high dose) tandem CAR5 T cells, anti-BCMA CAR T cells or anti-GPRC5D CAR T cells or mock-transduced T cells. Specific embodiments

[0095] The present application provides bispecific chimeric antigen receptors (CARs) (also referred to as "bispecific" CARs) that target or are directed against G protein-coupled receptor class C group 5 member D (GPRC5D) and B cell maturation antigen (BCMA). In some embodiments, the bispecific CARs provided by the present application target or are directed against cells and diseases that express GPRC5D and / or BCMA. The present application provides cells (such as T cells) that express the provided bispecific CARs and compositions containing such cells. It has been observed that GPRC5D is expressed, for example, heterogeneously, in certain diseases and disorders, such as malignancies, or on their tissues or cells, such as on malignant plasma cells from patients with relapsed or newly diagnosed myeloma, and is expressed at very low levels on normal tissues, for example. Among the provided embodiments are methods useful for treating diseases and disorders and / or for targeting such cell types, including nucleic acid molecules encoding a GPRC5D-binding binding domain and a BCMA-binding domain (including chimeric antigen receptors (CARs)), and the encoded receptors (such as the encoded CAR), and compositions and articles containing the same. These receptors typically may 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 scFv). The present application also provides cells, such as genetically engineered or recombinant cells that express such GPRC5D and BCMA-binding receptors (such as bispecific CARs) and / or contain nucleic acids encoding such receptors, and compositions, articles, and therapeutic doses containing such cells.

[0096] Embodiments provided by the present application relate to CAR T cells that target both GPRC5D and BCMA for the treatment of multiple myeloma. GPRC5D (Uniprot accession number Q9NZD1, as shown, for example, in SEQ ID NO:49) is a member of the G protein-coupled receptor class C group 5 member D belonging to the RAIG (retinoic acid-inducible gene-1) family. It is a 39 kDa G protein-coupled receptor with seven transmembrane helices, with two reported isoforms, and the isoform differences occur at the intracellular C-terminus of the protein. Results herein show that GPRC5D is highly expressed in multiple myeloma and, overall, is expressed at low levels in most normal tissues. BCMA (Uniprot Acc. No. Q02223, as shown in SEQ ID NO:60) is a type III transmembrane protein expressed on mature B lymphocytes. After BCMA binds to its ligands (B cell activator (BAFF) or proliferation-inducing ligand (APRIL) of the TNF family), a pro-survival cell signal is delivered to B cells, and this signal is found 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, leading to 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, and clinical efforts have mainly focused on targeting B cell maturation antigen (BCMA). In fact, there have been some recent advancements in the treatment options for MM, including two FDA-approved chimeric antigen receptor (CAR) T cell therapies targeting B cell maturation antigen (BCMA). However, although BCMA is expressed on many malignant plasma cells, in some cases, the expression levels can be diverse. In some aspects, the heterogeneity of target antigen expression can cause variable or inconsistent responses. In some aspects, it has also been observed that the expression of BCMA on the cell surface changes over time due to γ-secretase-mediated extracellular domain shedding. While some clinical trials have demonstrated high overall response rates, most patients will ultimately relapse, and a decrease in 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 a second antigen in MM can overcome antigen downregulation or loss, thereby reducing the opportunity for immune escape. For example, both BCMA and GPRC5D are highly expressed in MM, but their expression is independent of each other, making them a promising combination for dual targeting (Smith et al., Sci Transl Med (2019) 11(485):aau7746). Notably, the CAR provided in this application does not show obvious recombination (such as homologous recombination). Instead, due to the high sequence homology between different parts of the vector (e.g., parts encoding the same or similar components of each independent CAR), a dual-targeting CAR formatted in a bicistronic arrangement to allow the expression of two independent CARs from a single vector can exhibit unexpected or unwanted recombination. Lam et al., Blood (2021) 138(Suppl.1):4808.

[0098] In addition, in some cases, the recombinant receptor may exhibit antigen-independent activity or signal transduction (also known as "tonic signaling"), which can lead to undesirable effects, such as those caused by increased differentiation and / or exhaustion of T cells expressing the recombinant receptor. In some aspects, such activity may limit the activity, function, or efficacy of T cells. In some cases, during genetic engineering and ex vivo expansion of cells for recombinant receptor expression, the cells may exhibit a phenotype indicative of exhaustion due to tonic signaling induced by the recombinant receptor. In some cases, alternative or additional MM-targeted T cell therapies are needed.

[0099] In the engineered cells provided, the cells comprise a chimeric antigen receptor that exhibits high expression of a BCMA-binding domain and a GPRC5D-binding domain, as well as low tonic signal strength, thereby minimizing the likelihood of antigen-independent (tonic) signaling. In particular, the bispecific CARs provided herein include CARs having high antigen-dependent activation and minimal tonic signaling.

[0100] This application provides a single therapy regimen for targeting GPRC5D and BCMA expressed on autologous primary T cells using bispecific CARs, for use as a therapeutic agent for multiple myeloma plasma cells. In some embodiments, the single therapy regimen may be sufficient for a subject known or suspected or selected to have low or heterogeneous expression of BCMA on MM plasma cells. It has been observed that GPRC5D and BCMA are expressed, e.g., heterogeneously, on malignant plasma cells such as those from relapsed or newly diagnosed myeloma patients, and, for example, on certain diseases and conditions such as malignancies, or on their tissues or cells, while being expressed rarely on normal tissues. Due to the role of GPRC5D and BCMA in various diseases and disorders including cancer, GPRC5D and BCMA have become therapeutic targets.

[0101] In some cases, targeting two antigens as provided in this application may improve the depth and durability of response in patients while minimizing relapses caused by antigen escape. As demonstrated by data from CAR T cell trials in B cell malignancies, a mechanism of resistance to CAR T cell therapy can be loss or downregulation ("escape") of the target antigen. (Robbie G. Majzner and Crystal L. Mackall, Cancer Discov August 22 2018; DOI10.1158 / 2159-8290.CD-18-0442). Compared to therapies involving targeting only a single antigen, such dual-targeting strategies based on targeting two antigens can achieve synergistic or improved tumor responses. The dual-targeting approach can advantageously address issues caused by the likelihood of antigen loss and / or maximize antigen targeting in MM.

[0102] In addition, the CAR provided in this application shows strong functions against three different multiple myeloma cell lines in vitro and strong in vivo efficacy in three different multiple myeloma models, which demonstrates its adaptability in the presence of various antigen levels, up to and including the complete loss of antigen. To this end, the observations herein indicate that the provided CAR is highly functional when signaling through a single binding domain, which is consistent with the observation that CAR shows anti-tumor efficacy in the presence of only a single antigen (i.e., GPRC5D or BCMA), such as in the event of antigen loss.

[0103] The provided embodiments include methods useful for treating diseases and disorders and / or targeting such cell types, including nucleic acid molecules encoding bispecific chimeric antigen receptors (CARs) that bind both GPRC5D and BCMA, the encoded receptors (such as the encoded CAR), and compositions and articles comprising such receptors. The receptors generally may contain antibodies specific for GPRC5D and BCMA (including antigen-binding antibody fragments such as heavy chain variable regions, single domain antibody fragments, and single chain fragments, including single chain variable fragments (scFv)). In addition, cells are provided, such as engineered or recombinant cells expressing such CARs and / or containing nucleic acids encoding such receptors, and compositions, articles, and therapeutic doses comprising such cells.

[0104] All publications mentioned in this application, including patent documents, scientific papers, and databases, are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were incorporated by reference individually. If the definitions set forth in this application are contrary to or otherwise inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth in this application shall control.

[0105] The section headings used in this application are for organizational purposes only and should not be construed as limiting the subject matter described. I. Recombinant Receptors (such as Chimeric Antigen Receptors)

[0106] In some aspects, the present application provides GPRC5D and BCMA binders, such as recombinant receptors or chimeric antigen receptors (CARs), the binder comprising an extracellular binding domain that binds both GPRC5D and BCMA. The extracellular binding domain comprises a GPRC5D binding domain that binds GPRC5D and a BCMA binding domain that binds BCMA. The GPRC5D binding domain comprises a cell surface protein containing an antibody (such as an antigen-binding antibody fragment) and / or other binding peptides that specifically bind to GPRC5D (such as the human GPRC5D protein). The BCMA binding domain includes a cell surface protein containing an antibody (such as an antigen-binding antibody fragment) and / or other binding peptides that specifically bind to BCMA (such as human BCMA). In some aspects, the binding domain binds to the extracellular portion of GPRC5D. In some aspects, the GPRC5D binding domain binds to the extracellular portion of GPRC5D. In some aspects, the binding domain binds to the extracellular portion of BCMA. In some aspects, the BCMA binding domain binds to the extracellular portion of BCMA.

[0107] Among the provided polynucleotides are polynucleotides encoding recombinant receptors (such as antigen receptors) that specifically bind GPRC5D and BCMA. In some aspects, the encoded receptors are also provided, such as receptors containing GPRC5D and BCMA binding polypeptides, as well as compositions, articles, and uses thereof.

[0108] The GPRC5D and BCMA binding domains include antibodies, such as single-chain antibodies (such as antigen-binding antibody fragments) or portions thereof. In some instances, the recombinant receptor is a chimeric antigen receptor, such as those containing an anti-GPRC5D antibody or its antigen-binding fragment and an anti-BCMA antibody or its antigen-binding fragment, such as in tandem form. The provided polynucleotides can be integrated into constructs, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) constructs, for example, those that can be introduced into cells for the expression of the encoded recombinant GPRC5D and BCMA binding domains.

[0109] The provided recombinant receptors generally comprise 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 includes an extracellular binding domain comprising a GPRC5D binding domain and a BCMA binding domain. The recombinant receptor comprises a GPRC5D binding domain that specifically binds to GPRC5D, such as an anti-GPRC5D antibody, such as an antigen-binding fragment of GPRC5D. The recombinant receptor further includes a BCMA binding domain that specifically binds to BCMA, such as an anti-BCMA antibody, such as an antigen-binding fragment of BCMA. The antigen receptor is 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 for treating diseases and disorders associated with GPRC5D expression, BCMA expression, or both, such as multiple myeloma.

[0111] The chimeric receptor includes a chimeric antigen receptor (CAR). The CAR generally comprises an extracellular binding domain containing a GPRC5D binding domain and a BCMA binding domain, a transmembrane domain, and an intracellular signaling domain. The CAR generally further comprises a spacer sequence (such as containing a hinge sequence) between the extracellular binding domain and the transmembrane domain. Exemplary features of the CAR provided in this application will be further described below. 1. Extracellular antigen-binding domain

[0112] The chimeric receptor (such as a CAR) generally comprises an extracellular binding domain that includes, is, or comprises an anti-GPRC5D antibody and an anti-BCMA antibody. Thus, the chimeric receptor (such as a CAR) generally includes a GPRC5D binding domain and a BCMA binding domain in its extracellular portion, such as antigen-binding fragments, domains, or portions, 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 and an antigen-binding fragment thereof.

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

[0115] The terms "complementary determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art and refer to non-contiguous amino acid sequences within the variable region of an antibody that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art and refer to the non-CDR portions of the variable regions of the heavy and light chains. 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 exact amino acid sequence boundaries for a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in the following: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol, 1996; 262:732-745. ("Contact" numbering scheme); Lefranc MP et al., Dev Comp Immunol, 2003; 27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plückthun A, J Mol Biol, 2001; 309(3):657-70, ("Aho" numbering scheme); Martin et al., PNAS, 1989; 86(23):9268-9272, ("AbM" numbering scheme); and Ye et al., Nucleic Acids Res. 2013; 41(Web Server issue):W34-40, ("IgBLAST numbering scheme). Details of the various numbering schemes are described in the following publications: Jarasch et al., Proteins, 2017; 85(1):65-71; Martin et al., Bioinformatics tools for antibody engineering.In Dübel, S. (eds) Handbook of Therapeutic Antibodies, Vol. Wiley-VCH, Weinheim, Germany; Martin, A.C.R. (2010). Martin, A.C.R. (2010). Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg; and Martin, A.C.R., Antibody Information: How to identify the CDRs by looking at a sequence [online] bioinf.org.uk / abs / info.html, the above entire content is incorporated herein by reference in its entirety. Various predictive algorithm tools (such as AbYsis, Abnum, AbYmod, ABRSA, IgBLAST, IMGT or ANARCI) are available and are known to be used for numbering antibody residues and CDRs.

[0117] The boundaries given for CDR or FR can vary according to the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the length of the most common antibody region sequences, with insertions in some cases. Relative to the standard numbering scheme, insertions in the sequence are represented using insertion letter codes. For example, an insertion of a residue between residues L30 and L31 is represented as L31A, L31B, etc. Relative to the standard scheme, deletions in the sequence are represented by skipping numbers. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. For example, the Chothia numbering scheme is almost the same as the Kabat numbering scheme, except that insertions are located at structural positions and topologically equivalent residues are indeed given the same number. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects. The AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the definitions used in the AbM antibody modeling software of Oxford Molecular. The IgBLAST scheme is based on matching to germline V, D, and J genes and can be determined using the IgBLAST tool of the National Center for Biotechnology Information (NCBI) in the United States.

[0118] In some embodiments, Kabat numbering can be determined by known sequence rules, such as those 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 scheme can, in some respects, incorporate any of the following rules to label CDRs: CDR-L1 starts at approximately residue 24 of the light chain, always having a C residue before it and a W residue after it; the end of CDR-L1 is defined by a 3-residue extension, where the residue after the W residue can be Y, L, or F, followed by Q or L; the length of CDR-L1 is 10 to 17 residues; CDR-L2 always starts 16 residues after the end of CDR-L1; the two residues before CDR-L2 are I and Y, but can also be V and Y, I and K, or I and F; the length of CDR-L2 is always 7 residues; CDR-L3 always starts 33 residues after the end of CDR-L2, always having a C residue before it, followed by a strict F-G-X-G sequence motif, where X is any amino acid; the length of CDR-L3 is 7 to 11 residues; CDR-H1 starts at approximately position 26 of the heavy chain; the first amino acid in CDR-H1 is always 9 residues after a conserved C residue; there is an invariant W residue after CDR-H1, followed usually by V, but can also be I or A; the length of CDR-H1 is 5 to 7 residues; CDR-H2 always starts 15 residues after the end of CDR-H1; the first residue of CDR-H2 is usually preceded by an L-E-W-I-G sequence motif, but there are some variations; the end of CDR-H2 is defined by a 3-residue motif—the first residue of the 3-residue motif can be K or R, the second residue of the 3-residue motif can be L, I, V, F, T, or A, and the third residue of the 3-residue motif can be T, S, I, or A; the length of CDR-H2 is 16 to 19 residues; CDR-H3 always starts 33 residues after the end of CDR-H2 and is always 3 residues after a C residue—the first residue of CDR-H3 is preceded by a conserved C residue, followed by two residues, usually A-R; the residue after CDR-H3 strictly follows a W-G-X-G sequence motif, where X is any amino acid; the length of CDR-H3 is usually 3 to 25 residues; CDR-H3 can be much longer than 25 residues.

[0119] In some cases, according to the Chothia numbering scheme, the exact boundary positions of certain CDRs vary depending on the different definitions of CDRs (e.g., see Martin, ACR, Antibody Information: How to identify CDRs by looking at a sequence [online] bioinf.org.uk / abs / info.html). For example, in some cases, according to Chothia numbering, the boundary positions of CDR-L1 can be L26--L32 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M., J Mol Biol, 1987; 196(4):901-17). In some cases, the boundary positions of CDR-L1 may be L25--L32 (Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In certain cases, the boundary positions of CDR-L2 can be L50--L52, and the boundary positions of CDR-L3 can be L91--L96 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M., J Mol Biol, 1987; 196(4):901-17; and Al-Lazikani et al., J Mol Biol, 1997; 273(4):927-48). In some cases, according to Chothia numbering, the boundary positions of CDR-H1 can be H26--H32 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M., 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 positions of CDR-H2 can be H53--H55 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M., J Mol Biol, 1987; 196(4):901-17); 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 boundary positions of CDR-H3 can be H96--H101 (Chothia et al., Science, 1986; 233(4765):755-8; Chothia C. and Lesk A.M., J Mol Biol, 1987; 196(4):901-17). In certain cases, the boundary positions of CDR-H3 may be H92--H104 (Morea et al., 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 the exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, CDR-H3 identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbers are listed using both the Kabat and Chothia numbering schemes. The FRs are located between each CDR. 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, and so on. It should be noted that since the Kabat numbering scheme shown places insertions at H35A and H35B, depending on the loop length, the end of the Chothia CDR-H1 loop varies between H32 and H34 when numbered using the Kabat numbering convention shown. 1 - Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD 2 - Al-Lazikani et al., (1997) JMB 273, 927-948.

[0121] Accordingly, unless otherwise specified, the "CDR" or "complementary determining region" of a given antibody or its region (such as its variable region) or an individually specified CDR (e.g., CDR-H1, CDR-H2, CDR-H3) is understood to encompass the complementary determining regions as defined by any of the foregoing schemes or other known schemes. For example, in the case where it is stated that a particular CDR (e.g., CDR-H3) contains the amino acid sequence of the corresponding CDR in the amino acid sequence of a given VH or VL region, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the foregoing schemes or other known schemes.. In some embodiments, when it is stated that an antibody or its antigen-binding fragment contains CDR-H1, CDR-H2, and CDR-H3 contained in the amino acid sequence of a given VH region, and CDR-L1, CDR-L2, and CDR-L3 contained in the amino acid sequence of a given VL region, the CDRs can be defined by any of the foregoing schemes (such as the Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact schemes) or other known schemes. In some embodiments, specific CDR sequences are specified. The exemplary CDR sequences of the provided antibodies are described using various numbering schemes, but it is understood that the provided antibodies can include CDRs as described according to any of the other foregoing numbering schemes or other known numbering schemes.

[0122] Similarly, unless otherwise specified, the FR of a given antibody or its region (such as its variable region) or an individually specified FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4, FR-L1, FR-L2, FR-L3, and / or FR-L4) is understood to encompass the framework region (or a particular framework region) as defined by any known scheme. In some cases, the scheme used to identify a particular CDR, FR, or multiple FRs or CDRs, such as a CDR defined by the Kabat, Chothia, AbM, or Contact method or other known scheme, is specified. In other cases, the specific amino acid sequence of a CDR or FR is given. In some embodiments, when it is stated that an antibody or its antigen-binding fragment contains FR-H1, FR-H2, FR-H3, and FR-H4 contained in the amino acid sequence of a given VH region and FR-L1, FR-L2, FR-L3, and FR-L4 contained in the amino acid sequence of a given VL region, the FRs can be defined by any of the foregoing schemes (such as the Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method) or other known schemes.

[0123] The term "variable region" or "variable domain" refers to the domain in an antibody heavy or light chain that participates in antibody binding to an antigen. The heavy and light chains of a native antibody (V H and V L) variable regions generally have a similar structure, and each domain contains four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single V H or V L domain may be sufficient to confer antigen-binding specificity. In addition, V H or V L domains from an antibody that binds an antigen can be used to isolate antibodies that bind a specific antigen to screen libraries of complementary V L or V H domains, respectively. (See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0124] Antibody fragments are included among the provided antibodies. An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than a whole antibody that contains a portion of a whole antibody that binds the antigen to which the whole antibody binds. Examples of antibody fragments include (but are not limited to) Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; heavy chain variable (V H ) regions, single-chain antibody molecules such as scFv, and single-domain antibodies that contain only the VH region; and multispecific antibodies formed from antibody fragments. In some embodiments, the antibody is or includes an antibody fragment that contains a heavy chain variable (V H ) and a light chain variable (V L ) region. In certain embodiments, the antibody is a single-chain antibody fragment that contains a heavy chain variable (V H ) region and / or a light chain variable (VL) region, such as scFv.

[0125] A single-domain antibody (sdAb) is an antibody fragment that contains all or a portion of the heavy chain variable region of an antibody or all or a portion of the light chain variable region of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody

[0126] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of whole antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as one that contains fragments in a non-naturally occurring arrangement (such as those having two or more antibody regions or chains linked by a synthetic linker (e.g., a peptide linker)), and / or fragments that cannot be produced by enzymatic digestion of a naturally occurring whole antibody. In some aspects, the antibody fragment is scFv.

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

[0128] The provided antibodies include human antibodies. A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell or an antibody of non-human origin that utilizes a human antibody repertoire or other human antibody-encoding sequences (including a human antibody library). The term does not include humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those in which all or substantially all of the CDRs are non-human CDRs. The term includes antigen-binding fragments of human antibodies.

[0129] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies having human variable regions in response to antigen challenge. Such animals typically contain all or a portion of the human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin locus generally is not activated. Human antibodies also can be derived from human antibody libraries, including phage display libraries and cell-free libraries, that contain antibody-encoding sequences derived from the human repertoire.

[0130] The provided antibodies include monoclonal antibodies, including monoclonal antibody fragments. As used herein, the term "monoclonal antibody" refers to an antibody obtained from or within a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible variants that are present naturally or that arise during the preparation of the monoclonal antibody preparation, and these variants generally are present in trace amounts. Each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen, as compared to polyclonal antibody preparations that typically include different antibodies directed against different epitopes. The term should not be construed as requiring that the antibody be produced by any particular method. Monoclonal antibodies can be prepared by a variety of techniques, including (but not limited to) production from hybridomas, recombinant DNA methods, phage display, and other antibody display methods.

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

[0132] In some embodiments, the extracellular binding domain comprises a circular form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain 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.

[0133] In some embodiments, the extracellular binding domain comprises a circular form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain comprises: 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.

[0134] In some embodiments, the extracellular binding domain comprises a linear form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain comprises: 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; one of the VH and VL regions of the BCMA-binding domain; and the other of the VH and VL regions of the BCMA-binding domain.

[0135] In some embodiments, the extracellular binding domain comprises a linear form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain comprises: 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; one of the VH and VL regions of the GPRC5D-binding domain; and the other of the VH and VL regions of the GPRC5D-binding domain. a. GPRC5D-binding domain

[0136] In some embodiments, the GPRC5D-binding domain of the provided CAR contains an antibody, such as an anti-GPRC5D antibody, or an antigen-binding fragment thereof, and the antibody confers GPRC5D-binding properties to the provided CAR. In some embodiments, the CAR comprises a GPRC5D-binding domain containing an antibody (such as the heavy chain variable (VH) region and / or the light chain variable (VL) region of an antibody). In some embodiments, the (VH) and (VL) regions of the GPRC5D-binding domain are part of a tandem dual-targeting CAR having a BCMA-binding domain. 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 GPRC5D-binding domain comprise an scFv antibody fragment. In some embodiments, the antibody or antigen-binding domain can be any of the anti-GPRC5D antibodies described or derived from any of the anti-GPRC5D antibodies described (for example, see WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245). Any such anti-GPRC5D antibody or antigen-binding fragment can be used in the provided CAR. In some embodiments, the CAR comprises variable heavy (VH) and / or variable light (VL) regions derived from the antibodies described in the following patent documents: WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, or WO2018147245.

[0137] In some embodiments, the antibody (such as an anti-GPRC5D antibody) or antigen-binding fragment contains the VH or VL region sequences as described or a sufficiently antigen-binding portion thereof. In some embodiments, the anti-GPRC5D antibody (such as an antigen-binding fragment) comprises a V H region sequence containing CDR-H1, CDR-H2, and / or CDR-H3 as described or a sufficiently antigen-binding portion thereof. In some embodiments, the anti-GPRC5D antibody, such as an antigen-binding fragment, comprises a V LThe regional sequence or a sufficient antigen-binding portion thereof. In some embodiments, an anti-GPRC5D antibody, such as an antigen-binding fragment, comprises a VH region sequence containing CDR-H1, CDR-H2, and / or CDR-H3 as described, and comprises a VL region sequence containing CDR-L1, CDR-L2, and / or CDR-L3 as described. Additionally, the antibody includes antibodies having a sequence that is at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least about 99% identical to such a sequence.

[0138] In some embodiments, the antibody or antibody fragment in the provided CAR has the VH region of any of the antibodies or antibody-binding fragments described in WO2016 / 090312, WO2016 / 090329, WO2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245.

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

[0140] In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and / or CDR-H3 according to Kabat numbering. In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and / or CDR-H3 according to Chothia numbering. In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and / or CDR-H3 according to AbM numbering.

[0141] In some embodiments, the CAR comprises an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment thereof having a variable heavy chain (VH) region, the VH region comprising a CDR-H1 containing the amino acid sequence shown in SEQ ID NO:1, a CDR-H2 containing the amino acid sequence shown in SEQ ID NO:2, and a CDR-H3 containing the amino acid sequence shown in SEQ ID NO:3.

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

[0143] In some embodiments, the extracellular antigen-binding domain comprises a VH region containing the amino acid sequences shown in SEQ ID NOs: 1, 2, and 3.

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

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

[0146] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region containing the amino acid sequence shown in SEQ ID NO:7.

[0147] In some embodiments, the antibody or antibody fragment in the provided CAR comprising a VH 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 VH region and a VL region, or a sufficient antigen-binding portion of the VH and VL regions. In such embodiments, the VH region sequence can be any of the VH sequences described above. 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 further comprises a constant region.

[0148] In some embodiments, the CAR provided herein comprises an antibody, such as an anti-GPRC5D antibody, or an antigen-binding fragment thereof, that comprises any of the above VH regions and comprises a light chain variable region or a sufficient antigen-binding portion thereof. For example, in some embodiments, the CAR comprises an antibody or an antigen-binding fragment thereof that comprises a VH region and a light chain variable (VL) region, or a sufficient antigen-binding portion of the VH and VL regions. In such embodiments, the VH region sequence can be any of the VH sequences described above. 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 further comprises a constant region.

[0149] In some embodiments, the antibody or antigen-binding fragment has a VL region described in any of WO2016 / 090312, WO2016 / 090329, WO 2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245.

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

[0151] In some embodiments, the VL region of the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and / or CDR-L3 according to Kabat numbering. In some embodiments, the VL region of the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and / or CDR-L3 according to Chothia numbering. In some embodiments, the VL region of the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and / or CDR-L3 according to AbM numbering.

[0152] In certain embodiments, the CAR comprises an antibody or its antigen-binding fragment that has a variable light chain (VL) region that comprises CDR-L1 having the amino acid sequence shown in SEQ ID NO:4, CDR-L2 having the amino acid sequence shown in SEQ ID NO:5, and CDR-L3 having the amino acid sequence shown in SEQ ID NO:6.

[0153] In some embodiments, the antibody or its antigen-binding fragment comprises a VL region that comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences shown in SEQ ID NOs: 4, 5, and 6, respectively.

[0154] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 contained in the VH region amino acid sequence shown in SEQ ID NO:8, respectively.

[0155] The CARs provided in this application include such CARs, in which the antibody (such as an anti-GPRC5D antibody) or antibody fragment in the CARs provided herein comprises a VH region and a VL region, and the VH region has an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7, and the VL region comprises an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity with the amino acid shown in SEQ ID NO:8.

[0156] In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3, and the CDR-H1, CDR-H2, and CDR-H3 respectively comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained in the VH region amino acid sequence shown in SEQ ID NO:7; and comprises CDR-L1, CDR-L2, and CDR-L3, and the CDR-L1, CDR-L2, and CDR-L3 respectively comprise the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained in the VL region amino acid sequence shown in SEQ ID NO:8.

[0157] In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:7; and the VL region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the VH and VL regions of the antibody or its antigen-binding fragment respectively comprise the amino acid sequences shown in SEQ ID NO:7 and 8, or any antibody or its antigen-binding fragment having at least 90% sequence identity with any of the above VH and VL (e.g., having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith).

[0158] For example, the VH and VL regions of the antibody or its antigen-binding fragment provided herein respectively comprise the amino acid sequences shown in SEQ ID NO:7 and 8.

[0159] The provided CAR includes such a CAR in which the GPRC5D binding domain contains the amino acid sequences of the VH region and the VL region. The VH region contains the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7. The VL region contains the sequence shown in SEQ ID NO:8 or an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity with SEQ ID NO:8. In some embodiments, the GPRC5D binding domain of the provided CAR contains a VH region and a VL region. The VH region has CDRH1, CDRH2, and CDRH3 that respectively contain the amino acid sequences of SEQ ID NOs:1, 2, and 3. The VL region has CDRL1, CDRL2, and CDRL3 that respectively contain the amino acid sequences of SEQ ID NOs:4, 5, and 6. In some embodiments, the VH region contains the sequence shown in SEQ ID NO:7 and the VL region contains the sequence shown in SEQ ID NO:8.

[0160] In some embodiments, the GPRC5D-binding domain in the provided CAR is an antibody or an antigen-binding fragment thereof, which is a single-chain antibody fragment, such as a single-chain variable fragment (scFv), or a diabody, or a single-domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a single-domain antibody that contains only the VH region. In some embodiments, the antibody or antigen-binding fragment contains a heavy-chain variable (VH) region and a light-chain variable (VL) region. In some embodiments, the antibody or antigen-binding fragment is an scFv that contains a heavy-chain variable (VH) region and a light-chain variable (VL) region. In some embodiments, the single-chain antibody fragment (e.g., scFv) includes one or more linkers that connect two antibody domains or regions, such as the heavy-chain variable (VH) region and the light-chain variable (VL) region. The linker is typically a peptide linker, such as a flexible and / or soluble peptide linker. Among these linkers are those rich in glycine and serine and / or in some cases rich in threonine. In some embodiments, the linker further includes charged residues that can improve solubility, such as lysine and / or glutamate. In some embodiments, the linker further includes one or more prolines.

[0161] Thus, in some embodiments, the provided CAR contains an anti-GPRC5D antibody, which includes single-chain antibody fragments, such as scFv and diabodies, especially human single-chain antibody fragments, and typically contains a linker that connects two antibody domains or regions, such as the VH and VL regions. In some embodiments, the provided CAR contains an anti-BCMA antibody, which includes single-chain antibody fragments, such as scFv and diabodies, especially human single-chain antibody fragments, and typically contains a linker that connects two antibody domains or regions, such as the VH and VL regions. The linker is typically a peptide linker, such as a flexible and / or soluble peptide linker, such as a peptide linker rich in glycine and serine.

[0162] In some embodiments, the VH and VL region sequences of the GPRC5D-binding domain are sequentially linked by at least one spacer VH and VL region sequence of the BCMA-binding domain. In some embodiments, the extracellular antigen-binding domain of the CAR has a circular form, in which the VH and VL regions of the GPRC5D-binding domain are separated by the VH and VL regions of another BCMA-binding domain, forming a circular CAR. In some embodiments, at least one of the VH or VL region sequences of the GPRC5D-binding domain is directly linked to the VH and VL regions of the BCMA-binding domain through a linker.

[0163] In some embodiments, the CAR comprises a circular form. In some embodiments, the VH or VL region of the BCMA binding domain is linked to the VH or VL region of the GPRC5D binding domain via a linker. In some embodiments, one of the VH and VL regions of the BCMA binding domain is linked to one of the VH and VL regions of the BCMA binding domain via a linker. In some embodiments, one of the VH and VL regions of the GPRC5D binding domain is linked to the other of the VH and VL regions of the GPRC5D binding domain via a linker. In some embodiments, the linker is as shown in SEQ ID NO: 17. In some embodiments, the linker is as shown in SEQ ID NO: 18. In some embodiments, the linker is as shown in SEQ ID NO: 19. In some embodiments, the linker is as shown in SEQ ID NO: 21. In some embodiments, the linker is as shown in SEQ ID NO: 22.

[0164] In some embodiments, the VH region of the BCMA binding domain is linked to the VL region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 19. In some embodiments, the VH region of the BCMA binding domain is linked to the VL region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 21. In some embodiments, the VH region of the BCMA binding domain is linked to the VL region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 22. In some embodiments, the VH region of the BCMA binding domain is linked to the VH region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 19. In some embodiments, the VH region of the BCMA binding domain is linked to the VH region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 21. In some embodiments, the VH region of the BCMA binding domain is linked to the VH region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 22.

[0165] In some embodiments, the VL region of the BCMA-binding domain is linked to the VL region of the GPRC5D-binding domain by the linker shown in SEQ ID NO: 19. In some embodiments, the VL region of the BCMA-binding domain is linked to the VL region of the GPRC5D-binding domain by the linker shown in SEQ ID NO: 21. In some embodiments, the VL region of the BCMA-binding domain is linked to the VL region of the GPRC5D-binding domain by the linker shown in SEQ ID NO: 22. In some embodiments, the VL region of the BCMA-binding domain is linked to the VH region of the GPRC5D-binding domain by the linker shown in SEQ ID NO: 19. In some embodiments, the VL region of the BCMA-binding domain is linked to the VH region of the GPRC5D-binding domain by the linker shown in SEQ ID NO: 21. In some embodiments, the VL region of the BCMA-binding domain is linked to the VH region of the GPRC5D-binding domain by the linker shown in SEQ ID NO: 22.

[0166] In some embodiments, the extracellular antigen-binding domain of the CAR has a linear form, wherein the VH and VL regions of the GPRC5D-binding domain are directly linked in sequence by a linker (such as as an scFv), and the VH and VL regions of the BCMA-binding domain are directly linked in sequence by a linker (such as as an scFv). In some embodiments, the GPRC5D-binding domain comprises a linker between the VH and VL regions. In some embodiments, in the order from the N-terminus to the C-terminus, the GPRC5D-binding domain comprises one of the VH and VL regions, a linker, and the other of the VH and VL regions. In some embodiments, the linker is as shown in SEQ ID NO: 17. Thus, in some embodiments, in the order from the N-terminus to the C-terminus, the GPRC5D-binding domain comprises one of the VH and VL regions, the linker shown in SEQ ID NO: 17, and the other of the VH and VL regions.

[0167] In some aspects, 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 or at least about 50%, 55%, 60%, 70% or 75% glycine, serine and / or threonine. In some embodiments, the linker consists essentially of glycine, serine and / or threonine. The length of the linker is typically between about 5 and about 50 amino acids, typically between or about 10 and or about 30 amino acids, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids, and in some instances between 10 and 25 amino acids in length. Exemplary linkers include linkers having sequences with various numbers of repeats of GGGGS (4GS; SEQ ID NO: 21) or GGGS (3GS; SEQ ID NO: 20), such as between 2, 3, 4 and 5 repeats of such sequences. Exemplary linkers include or consist of linkers having the sequences shown in SEQ ID NO: 22 (GGGGSGGGGS), SEQ ID NO: 23 (GGGGSGGGGSGGGS) and SEQ ID NO: 24 (GGGGSGGGGSGGGGS). Exemplary linkers also include or consist of linkers having the sequences shown in SEQ ID NO: 18 (GSTSGSGKPGSGEGSTKG), SEQ ID NO: 17 (GSRGGGGSGGGGSGGGSLEMA) and SEQ ID NO: 19 (EAAAK).

[0168] Thus, in some embodiments, the provided embodiments include single-chain antibody fragments, such as scFv, which comprise one or more of the foregoing linkers, such as glycine / serine-rich linkers, including linkers having repeats of GGGS (SEQ ID NO:20) or GGGGS (SEQ ID NO:21), such as the linkers shown in SEQ ID NO:17, 18, 19, 22, 23, or 24. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:17. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:18. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:19. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:20. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:21. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:22. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:23. In some embodiments, the linker comprises the sequence shown in SEQ ID NO:24.

[0169] In some embodiments, the VH region can be the amino terminus of the VL region. In some embodiments, the VH region can be the carboxyl terminus of the VL region. In certain embodiments, the fragment (such as scFv) can comprise the VH region or a portion thereof, followed by a linker, followed by the VL region or a portion thereof. In other embodiments, the fragment (such as scFv) can comprise the VL region or a portion thereof, followed by a linker, followed by the VH region or a portion thereof.

[0170] In some embodiments, the extracellular binding domain comprises a linear form. 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 as shown in SEQ ID NO:19. In some embodiments, the linker is as shown in SEQ ID NO:20. In some embodiments, the linker is as shown in SEQ ID NO:24. In some embodiments, the VH and VL regions of the anti-GPRC5D scFv are linked by the linker shown in SEQ ID NO:17. In some embodiments, the VH and VL regions of the anti-BCMA scFv are linked by the linker shown in SEQ ID NO:17.

[0171] In some aspects, the scFv provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46, or an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98% or at least or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 45 or SEQ ID NO: 46. In some aspects, the scFv provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 45, or an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98% or at least or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 45. In some aspects, the scFv provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 45. In some embodiments, the scFv provided herein comprises an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98% or at least or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 45. In some aspects, the scFv provided by the present application comprises the amino acid sequence shown in SEQ ID NO: 46, or an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98% or at least or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 46. In some aspects, the scFv provided herein comprises the amino acid sequence shown in SEQ ID NO: 46. In some embodiments, the scFv provided herein comprises an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98% or at least or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 46.

[0172] The antibodies in the provided CARs, such as antigen-binding fragments, include human antibodies. In some embodiments of the provided human anti-GPRC5D antibodies, such as antigen-binding fragments, the human antibody contains a VH region 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; and / or contains a VL region 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 κ or λ chain V 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 κ or λ chain J segment. In some embodiments, this portion of the VH region corresponds to CDR-H1, CDR-H2, and / or CDR-H3. In some embodiments, this portion of the VH region corresponds to framework region 1 (FR1), FR2, FR2, and / or FR4. In some embodiments, this portion of the VL region corresponds to CDR-L1, CDR-L2, and / or CDR-L3. In some embodiments, this portion of the VL region corresponds to FR1, FR2, FR2, and / or FR4.

[0173] In some embodiments, the human antibody, such as an antigen-binding fragment, contains a CDR-H1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H1 region within the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, the CDR-H1 contained in the human antibody has a sequence that is 100% identical to the corresponding CDR-H1 region within the sequence encoded by a germline nucleotide human heavy chain V segment, or has no more than one, two, or three amino acid differences therefrom.

[0174] In some embodiments, the human antibody, such as an antigen-binding fragment, contains a CDR-H2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H2 region within the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, the CDR-H2 contained in the human antibody has a sequence that is 100% identical to the corresponding CDR-H2 region within the sequence encoded by a germline nucleotide human heavy chain V segment, or has no more than one, two, or three amino acid differences therefrom.

[0175] In some embodiments, a human antibody, such as an antigen-binding fragment, comprises a CDR-H3 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-H3 region within the sequence encoded by the germline nucleotide human heavy chain V segment, D segment, and J segment. For example, in some embodiments, a human antibody comprises a CDR-H3 having 100% identity to the corresponding CDR-H3 region within the sequence encoded by the germline nucleotide human heavy chain V segment, D segment, and J segment, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0176] In some embodiments, a human antibody, such as an antigen-binding fragment, comprises a CDR-L1 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L1 region within the sequence encoded by the germline nucleotide human light chain V segment. For example, in some embodiments, a human antibody comprises a CDR-L1 having 100% identity to the corresponding CDR-L1 region within the sequence encoded by the germline nucleotide human light chain V segment, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0177] In some embodiments, a human antibody, such as an antigen-binding fragment, comprises a CDR-L2 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L2 region within the sequence encoded by the germline nucleotide human light chain V segment. For example, in some embodiments, a human antibody comprises a CDR-L2 having 100% identity to the corresponding CDR-L2 region within the sequence encoded by the germline nucleotide human light chain V segment, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0178] In some embodiments, a human antibody, such as an antigen-binding fragment, comprises a CDR-L3 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L3 region within the sequence encoded by the germline nucleotide human light chain V segment and J segment. For example, in some embodiments, a human antibody comprises a CDR-L3 having 100% identity to the corresponding CDR-L3 region within the sequence encoded by the germline nucleotide human light chain V segment and J segment, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0179] In some embodiments, a human antibody, such as an antigen-binding fragment, comprises a framework region that contains human germline gene segment sequences. For example, in some embodiments, a human antibody contains a VH region in which the framework regions, such as FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the framework regions encoded by human germline antibody segments, such as V segments and / or J segments. In some embodiments, a human antibody contains a VL region in which the framework regions, such as FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the framework regions encoded by human germline antibody segments, such as V segments and / or J segments. For example, in some such embodiments, the framework region sequences contained within the VH region and / or VL region have no more than 10 amino acid differences, such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference, from the framework region sequences encoded by human germline antibody segments. b. BCMA-binding domain

[0180] In some embodiments, the BCMA-binding domain of the provided CAR contains an antibody, such as an anti-BCMA antibody, or an antigen-binding fragment thereof, which confers the BCMA-binding properties of the provided CAR. In some embodiments, the CAR comprises a BCMA-binding domain that contains an antibody (such as the heavy-chain variable (VH) region and / or the light-chain variable (VL) region of an antibody). In some embodiments, the (VH) and (VL) regions of the BCMA-binding domain are part of a tandem dual-targeting CAR that has a BCMA-binding domain. In some embodiments, 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 BCMA-binding domain contain an scFv antibody fragment. In some embodiments, the antibody or antigen-binding domain can be any of the anti-BCMA antibodies described or derived from any of the anti-BCMA antibodies described (see, e.g., WO2016 / 090320 or WO2016 / 090327). Any such anti-BCMA antibody or antigen-binding fragment can be used in the provided CAR. In some embodiments, the CAR comprises a variable heavy (VH) region and / or a variable light (VL) region derived from an antibody described in WO 2016 / 090320 or WO2016 / 090327.

[0181] In some embodiments, an antibody, such as an anti-BCMA antibody, or an antigen-binding fragment contains a heavy and / or light chain variable (VH or VL) region sequence as described or a sufficient antigen-binding portion thereof. In some embodiments, an anti-BCMA antibody, such as an antigen-binding fragment, includes a VH region sequence containing CDR-H1, CDR-H2, and / or CDR-H3 as described or a sufficient antigen-binding portion thereof. In some embodiments, an anti-BCMA antibody, such as an antigen-binding fragment, includes a VL region sequence containing CDR-L1, CDR-L2, and / or CDR-L3 as described or a sufficient antigen-binding portion thereof. In some embodiments, an anti-BCMA antibody, such as an antigen-binding fragment, includes a VH region sequence containing CDR-H1, CDR-H2, and / or CDR-H3 as described and includes a VL region sequence containing CDR-L1, CDR-L2, and / or CDR-L3 as described. Also included in the antibodies are antibodies having a sequence that is at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least about 99% identical to such sequences.

[0182] In some embodiments, the antibody or antibody fragment in the provided CAR has a VH region of any of the antibodies or antibody-binding fragments described in either WO2016 / 090320 or WO2016 / 090327.

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

[0184] In some embodiments, the VH region of the antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and / or CDR-H3 according to Kabat numbering. In some embodiments, the VH region of the antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and / or CDR-H3 according to Chothia numbering. In some embodiments, the VH region of the antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and / or CDR-H3 according to AbM numbering.

[0185] In certain embodiments, the CAR comprises an antibody or antigen-binding fragment thereof having a variable heavy chain (VH) region that comprises CDR-H1 of the amino acid sequence shown in SEQ ID NO:9, CDR-H2 of the amino acid sequence shown in SEQ ID NO:10, and CDR-H3 of the amino acid sequence shown in SEQ ID NO:11.

[0186] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region that comprises CDR-H1, CDR-H2, and CDR-H3 that contain the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11, respectively.

[0187] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region that contains the amino acid sequences shown in SEQ ID NOs: 9, 10, and 11.

[0188] In some embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR-H3 that respectively comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence shown in SEQ ID NO:15.

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

[0190] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH region, and the VH region comprises the amino acid sequence shown in SEQ ID NO: 15.

[0191] In some embodiments, the antibody or antibody fragment in the CAR comprising a VH 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 VH region and a VL region, or a sufficient antigen-binding portion of the VH region and the VL region. In such embodiments, the VH region sequence can be any of the VH sequences described above. In some 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 further comprises a constant region.

[0192] In some embodiments, the antibody (such as an anti-BCMA antibody) or antigen-binding fragment thereof in the CAR provided herein comprises any of the above VH regions and comprises a variable light chain region or a sufficient antigen-binding portion thereof. For example, in some embodiments, the CAR comprises an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof comprises a VH region and a variable light chain (VL) region, or a sufficient antigen-binding portion of the VH region and the VL region. In such embodiments, the VH region sequence can be any of the VH sequences described above. In some 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 further comprises a constant region.

[0193] In some embodiments, the antibody or antigen-binding fragment has a VL region as described in any one of WO2016 / 090320 or WO2016 / 090327.

[0194] In some embodiments, the CAR comprises an antibody or antigen-binding fragment thereof, and the antibody or antigen-binding fragment thereof has a variable light chain (VL) region, and the variable light chain (VL) region has the amino acid sequence shown in SEQ ID NO: 16, or an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98% or at least or at least about 99% sequence identity to the VL region amino acids shown in SEQ ID NO: 16, or comprises CDR-L1, CDR-L2, and / or CDR-L3 present in such VL sequences.

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

[0196] In certain embodiments, the CAR comprises an antibody or antigen-binding fragment thereof having a variable light (VL) region that comprises CDR-L1 having the amino acid sequence set forth in SEQ ID NO:12, CDR-L2 having the amino acid sequence set forth in SEQ ID NO:13, and CDR-L3 having the amino acid sequence set forth in SEQ ID NO:14.

[0197] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL region that comprises CDR-L1, CDR-L2, and CDR-L3 having the amino acid sequences set forth in SEQ ID NO:12, 13, and 14, respectively.

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

[0199] The provided CARs include such CARs in which the antibody (such as an anti-BCMA antibody) or antibody fragment in the CAR comprises a VH region and a VL region, the VH region comprising an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15, and the VL region comprising an amino acid sequence having at least or at least about 90%, at least or at least about 91%, at least or at least about 92%, at least or at least about 93%, at least or at least about 94%, at least or at least about 95%, at least or at least about 96%, at least or at least about 97%, at least or at least about 98%, or at least or at least about 99% sequence identity to the amino acid set forth in SEQ ID NO:16.

[0200] In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3, and the CDR-H1, CDR-H2, and CDR-H3 respectively comprise the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the VH region amino acid sequence shown in SEQ ID NO:15; and comprises CDR-L1, CDR-L2, and CDR-L3, and the CDR-L1, CDR-L2, and CDR-L3 respectively comprise the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained within the VL region amino acid sequence shown in SEQ ID NO:16.

[0201] In some embodiments, the VH region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:15; and the VL region of the antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:16. In some embodiments, the VH and VL regions of the antibody or its antigen-binding fragment respectively comprise the amino acid sequences shown in SEQ ID NO:15 and 16, or any antibody or its antigen-binding fragment having at least 90% sequence identity with any of the above VH and VL, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[0202] For example, the VH and VL regions of the antibody or its antigen-binding fragment provided herein respectively comprise the amino acid sequences shown in SEQ ID NO:15 and 16.

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

[0204] In some embodiments, the BCMA-binding domain in the provided CAR is an antibody or an antigen-binding fragment thereof, which is a single-chain antibody fragment, such as a single-chain variable fragment (scFv), or a diabody, or a single-domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a single-domain antibody that only contains the VH region. In some embodiments, the antibody or antigen-binding fragment contains a heavy-chain variable (VH) region and a light-chain variable (VL) region. In some embodiments, the antibody or antigen-binding fragment is an scFv that contains a heavy-chain variable (VH) region and a light-chain variable (VL) region. In some embodiments, the single-chain antibody fragment (e.g., scFv) includes one or more linkers that connect two antibody domains or regions, such as the heavy-chain variable (VH) region and the light-chain variable (VL) region. The linker is typically a peptide linker, such as a flexible and / or soluble peptide linker. Among these linkers are those rich in glycine and serine and / or in some cases rich in threonine. In some embodiments, the linker further includes charged residues that can improve solubility, such as lysine and / or glutamate. In some embodiments, the linker further includes one or more prolines.

[0205] Thus, in some embodiments, the provided CAR contains an anti-BCMA antibody, which includes single-chain antibody fragments, such as scFv and diabodies, especially human single-chain antibody fragments, and typically contains a linker that connects two antibody domains or regions, such as the VH and VL regions. The linker is typically a peptide linker, such as a flexible and / or soluble peptide linker, such as a peptide linker rich in glycine and serine.

[0206] In some embodiments, the VH and VL region sequences of the BCMA-binding domain are sequentially connected by at least one spacer VH and VL region sequence of the GPRC5D-binding domain. In some embodiments, the extracellular antigen-binding domain of the CAR has a circular form, in which the VH and VL regions of the BCMA-binding domain are spaced by the VH and VL regions of another GPRC5D-binding domain to form a circular CAR. In some embodiments, at least one of the VH or VL region sequences of the BCMA-binding domain is directly connected to the VH and VL regions of the GPRC5D-binding domain through a linker.

[0207] In some embodiments, the CAR comprises a circular form. In some embodiments, the VH or VL region of the BCMA binding domain is linked to the VH or VL region of the GPRC5D binding domain via a linker. In some embodiments, one of the VH and VL regions of the BCMA binding domain is linked to one of the VH and VL regions of the GPRC5D binding domain via a linker. In some embodiments, one of the VH and VL regions of the GPRC5D binding domain is linked to the other of the VH and VL regions of the GPRC5D binding domain via a linker. In some embodiments, the linker is as shown in SEQ ID NO: 17. In some embodiments, the linker is as shown in SEQ ID NO: 18. In some embodiments, the linker is as shown in SEQ ID NO: 19. In some embodiments, the linker is as shown in SEQ ID NO: 21. In some embodiments, the linker is as shown in SEQ ID NO: 22.

[0208] In some embodiments, the VH region of the BCMA binding domain is linked to the VL region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 19. In some embodiments, the VH region of the BCMA binding domain is linked to the VL region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 21. In some embodiments, the VH region of the BCMA binding domain is linked to the VL region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 22. In some embodiments, the VH region of the BCMA binding domain is linked to the VH region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 19. In some embodiments, the VH region of the BCMA binding domain is linked to the VH region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 21. In some embodiments, the VH region of the BCMA binding domain is linked to the VH region of the GPRC5D binding domain via the linker as shown in SEQ ID NO: 22.

[0209] In some embodiments, the VL region of the BCMA-binding domain is linked to the VL region of the GPRC5D-binding domain via the linker shown in SEQ ID NO: 19. In some embodiments, the VL region of the BCMA-binding domain is linked to the VL region of the GPRC5D-binding domain via the linker shown in SEQ ID NO: 21. In some embodiments, the VL region of the BCMA-binding domain is linked to the VL region of the GPRC5D-binding domain via the linker shown in SEQ ID NO: 22. In some embodiments, the VL region of the BCMA-binding domain is linked to the VH region of the GPRC5D-binding domain via the linker shown in SEQ ID NO: 19. In some embodiments, the VL region of the BCMA-binding domain is linked to the VH region of the GPRC5D-binding domain via the linker shown in SEQ ID NO: 21. In some embodiments, the VL region of the BCMA-binding domain is linked to the VH region of the GPRC5D-binding domain via the linker shown in SEQ ID NO: 22.

[0210] In some embodiments, the extracellular antigen-binding domain of the CAR has a linear form, wherein the VH and VL regions of the BCMA-binding domain are directly linked in sequence via a linker (such as as an scFv), and the VH and VL regions of the GPRC5D-binding domain are directly linked in sequence via a linker (such as as an scFv). In some embodiments, the BCMA-binding domain comprises a linker between the VH and VL regions. In some embodiments, in the order from the N-terminus to the C-terminus, the BCMA-binding domain comprises one of the VH and VL regions, a linker, and the other of the VH and VL regions. In some embodiments, the linker is as shown in SEQ ID NO: 17. Thus, in some embodiments, in the order from the N-terminus to the C-terminus, the BCMA-binding domain comprises one of the VH and VL regions, the linker shown in SEQ ID NO: 17, and the other of the VH and VL regions.

[0211] In some aspects, glycine- and serine- (and / or threonine-) rich linkers contain at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of such amino acids. In some embodiments, they contain at least or at least about 50%, 55%, 60%, 70% or 75% glycine, serine and / or threonine. In some embodiments, the linker consists essentially of glycine, serine and / or threonine. The length of the linker is typically between about 5 and about 50 amino acids, typically between or about 10 and or about 30 amino acids, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids, and in some instances between 10 and 25 amino acids in length. Exemplary linkers include linkers having various numbers of repeats of GGGGS (4GS; SEQ ID NO: 21) or GGGS (3GS; SEQ ID NO: 20), such as between 2, 3, 4 and 5 repeats of such sequences. Exemplary linkers include linkers having or consisting of the sequences shown in SEQ ID NO: 22 (GGGGSGGGGS), SEQ ID NO: 23 (GGGGSGGGGSGGGS) and SEQ ID NO: 24 (GGGGSGGGGSGGGGS). Exemplary linkers also include linkers having or consisting of the sequences shown in SEQ ID NO: 18 (GSTSGSGKPGSGEGSTKG), SEQ ID NO: 17 (GSRGGGGSGGGGSGGGSLEMA) and SEQ ID NO: 19 (EAAAK).

[0212] Thus, in some embodiments, the provided embodiments include single-chain antibody fragments, such as scFv, which contain one or more of the foregoing linkers, such as glycine / serine-rich linkers, including linkers having repeats of GGGS (SEQ ID NO:20) or GGGGS (SEQ ID NO:21), such as the linkers shown in SEQ ID NO:17, 18, 19, 22, 23, or 24. In some embodiments, the linker contains the sequence shown in SEQ ID NO:17. In some embodiments, the linker contains the sequence shown in SEQ ID NO:18. In some embodiments, the linker contains the sequence shown in SEQ ID NO:19. In some embodiments, the linker contains the sequence shown in SEQ ID NO:20. In some embodiments, the linker contains the sequence shown in SEQ ID NO:21. In some embodiments, the linker contains the sequence shown in SEQ ID NO:22. In some embodiments, the linker contains the sequence shown in SEQ ID NO:23. In some embodiments, the linker contains the sequence shown in SEQ ID NO:24.

[0213] In some embodiments, the VH region can be the amino terminus of the VL region. In some embodiments, the VH region can be the carboxyl terminus of the VL region. In certain embodiments, the fragment (such as scFv) can contain the VH region or a portion thereof, followed by a linker, followed by the VL region or a portion thereof. In other embodiments, the fragment (such as scFv) can contain the VL region or a portion thereof, followed by a linker, followed by the VH region or a portion thereof.

[0214] In some embodiments, the extracellular binding domain comprises a linear form. 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 as shown in SEQ ID NO:19. In some embodiments, the linker is as shown in SEQ ID NO:21. In some embodiments, the linker is as shown in SEQ ID NO:24. In some embodiments, the VH and VL regions of the anti-GPRC5D scFv are linked by the linker shown in SEQ ID NO:17. In some embodiments, the VH and VL regions of the anti-BCMA scFv are linked by the linker shown in SEQ ID NO:17.

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

[0216] The antibodies in the provided CARs, such as antigen-binding fragments, include human antibodies. In some embodiments of the provided human anti-BCMA antibodies, such as antigen-binding fragments, the human antibody contains a VH region 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; and / or contains a VL region 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 κ or λ chain V 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 κ or λ chain J segment. In some embodiments, this portion of the VH region corresponds to CDR-H1, CDR-H2, and / or CDR-H3. In some embodiments, this portion of the VH region corresponds to framework region 1 (FR1), FR2, FR2, and / or FR4. In some embodiments, this portion of the VL region corresponds to CDR-L1, CDR-L2, and / or CDR-L3. In some embodiments, this portion of the VL region corresponds to FR1, FR2, FR2, and / or FR4.

[0217] In some embodiments, the human antibody, such as an antigen-binding fragment, contains a CDR-H1 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H1 region within the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, the CDR-H1 contained in the human antibody has a sequence that is 100% identical to the corresponding CDR-H1 region within the sequence encoded by a germline nucleotide human heavy chain V segment, or has no more than one, two, or three amino acid differences therefrom.

[0218] In some embodiments, the human antibody, such as an antigen-binding fragment, contains a CDR-H2 having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the corresponding CDR-H2 region within the sequence encoded by a germline nucleotide human heavy chain V segment. For example, in some embodiments, the CDR-H2 contained in the human antibody has a sequence that is 100% identical to the corresponding CDR-H2 region within the sequence encoded by a germline nucleotide human heavy chain V segment, or has no more than one, two, or three amino acid differences therefrom.

[0219] In some embodiments, a human antibody, such as an antigen-binding fragment, contains a CDR-H3 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-H3 region within the sequence encoded by the germline nucleotide human heavy chain V, D, and J regions. For example, in some embodiments, the human antibody contains a CDR-H3 having 100% identity to the corresponding CDR-H3 region within the sequence encoded by the germline nucleotide human heavy chain V, D, and J regions, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0220] In some embodiments, a human antibody, such as an antigen-binding fragment, contains a CDR-L1 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L1 region within the sequence encoded by the germline nucleotide human light chain V region. For example, in some embodiments, the human antibody contains a CDR-L1 having 100% identity to the corresponding CDR-L1 region within the sequence encoded by the germline nucleotide human light chain V region, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0221] In some embodiments, a human antibody, such as an antigen-binding fragment, contains a CDR-L2 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L2 region within the sequence encoded by the germline nucleotide human light chain V region. For example, in some embodiments, the human antibody contains a CDR-L2 having 100% identity to the corresponding CDR-L2 region within the sequence encoded by the germline nucleotide human light chain V region, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0222] In some embodiments, a human antibody, such as an antigen-binding fragment, contains a CDR-L3 having at least 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of the corresponding CDR-L3 region within the sequence encoded by the germline nucleotide human light chain V and J regions. For example, in some embodiments, the human antibody contains a CDR-L3 having 100% identity to the corresponding CDR-L3 region within the sequence encoded by the germline nucleotide human light chain V and J regions, or a sequence having no more than one, two, or three amino acid differences therefrom.

[0223] In some embodiments, a human antibody, such as an antigen-binding fragment, includes a framework region containing human germline gene segment sequences. For example, in some embodiments, a human antibody contains a VH region in which the framework regions, such as FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the framework regions encoded by human germline antibody segments, such as V segments and / or J segments. In some embodiments, a human antibody contains a VL region in which the framework regions, such as FR1, FR2, FR3, and FR4, have at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the framework regions encoded by human germline antibody segments, such as V segments and / or J segments. For example, in some such embodiments, the framework region sequences contained within the VH region and / or VL region have no more than 10 amino acid differences, such as no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid difference, from the framework region sequences encoded by human germline antibody segments. c. Exemplary dual-targeting extracellular antigen-binding domains

[0224] In some embodiments, the extracellular binding domain comprises a circular form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain comprises: 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. In some embodiments, the extracellular binding domain comprises a domain linker (e.g., a first and a second domain linker) that separates the VH or VL region of the GPRC5D binding domain from the VH or VL region of the BCMA binding domain. In some embodiments, there are a first and a second domain linker, and the linkers are the same. In some embodiments, the linker is any of those described herein. In some embodiments, the domain linker is shown as any one of SEQ ID NO: 19, 21, 22, or 24. In some embodiments, the extracellular binding domain comprises an intradomain linker that separates the VH and VL regions of the BCMA binding domain. In some embodiments, the intradomain linker is any of those described herein. In some embodiments, the intradomain linker is shown as SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO: 83 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 83. In some embodiments, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO: 84 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 84. In some embodiments, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO: 87 or an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 87. In some such embodiments, the extracellular antigen-binding domain targets the binding of the CAR for dual targeting of GPRC5D and BCMA.

[0225] In some embodiments, the extracellular binding domain comprises a circular form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain 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. In some embodiments, the extracellular binding domain comprises a domain linker (e.g., a first and a second domain linker) that separates the VH or VL region of the BCMA binding domain from the VH or VL region of the GPRC5D binding domain. In some embodiments, the linker is any of those described herein. In some embodiments, there are a first and a second domain linker, and the linkers are the same. In some embodiments, the domain linker is any one of SEQ ID NO: 19, 21, 22, or 24. In some embodiments, the extracellular binding domain comprises an intradomain linker that separates the VH and VL regions of the GPRC5D binding domain. In some embodiments, the intradomain linker is any of those described herein. In some embodiments, the intradomain linker is as shown in SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO: 81 or an amino acid sequence having 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 the amino acid sequence shown in SEQ ID NO: 82 or an amino acid sequence having 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 the amino acid sequence shown in SEQ ID NO: 85 or an amino acid sequence having 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 the amino acid sequence shown in SEQ ID NO: 86 or an amino acid sequence having 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 the amino acid sequence shown in SEQ ID NO:88 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:88. In some embodiments, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO:89 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:89. In some embodiments, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO:90 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:90. In some such embodiments, the extracellular antigen-binding domain targets the binding of the CAR for dual targeting of GPRC5D and BCMA.

[0226] In some embodiments, the extracellular binding domain comprises a linear form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain comprises: 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; one of the VH and VL regions of the BCMA binding domain; and the other of the VH and VL regions of the BCMA binding domain. In some embodiments, the extracellular binding domain comprises an intra-domain linker that separates the VH and VL regions of the GPRC5D binding domain. In some embodiments, the extracellular binding domain comprises an intra-domain linker that separates the VH and VL regions of the BCMA binding domain. In some embodiments, the intra-domain linker is any of those described herein. In some embodiments, the intra-domain linker is as set forth in SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the extracellular binding domain comprises an inter-domain linker that separates the VH or VL region of the GPRC5D binding domain from the VH or VL region of the BCMA binding domain. In some embodiments, the linker is any of those described herein. In some embodiments, there are first and second inter-domain linkers and the linkers are the same. In some embodiments, the inter-domain linker is as set forth in any of SEQ ID NO: 19, 21, 22 or 24. In some embodiments, the extracellular binding domain has the amino acid sequence set forth in SEQ ID NO: 77 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 77. In some embodiments, the extracellular binding domain has the amino acid sequence set forth in SEQ ID NO: 78 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 78. In some embodiments, the extracellular binding domain has the amino acid sequence set forth in SEQ ID NO: 79 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 79.In some embodiments, the extracellular binding domain has the amino acid sequence shown in SEQ ID NO:80 or an amino acid 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:80. In some such embodiments, the extracellular antigen-binding domain targets the binding of the CAR for dual targeting of GPRC5D and BCMA.

[0227] In some embodiments, the extracellular binding domain comprises a linear form. In some embodiments, from the N-terminus to the C-terminus, the extracellular binding domain comprises: 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; one of the VH and VL regions of the GPRC5D-binding domain; and the other of the VH and VL regions of the GPRC5D-binding domain. In some such embodiments, the extracellular antigen-binding domain targets the binding of the CAR for dual targeting of GPRC5D and BCMA.

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

[0229] In some embodiments, the extracellular binding domain comprises the amino acid sequence shown 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 shown in SEQ ID NO: 77. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO: 78. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO: 79. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO: 80. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO: 83. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO: 84. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO: 87.

[0230] In some embodiments, the extracellular binding domain is configured to bring the GPRC5D targeting binding domain closer to the transmembrane domain.

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

[0232] In some embodiments, the extracellular binding domain is configured to bring the BCMA targeting binding domain closer to the transmembrane domain.

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

[0234] In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in any one of SEQ ID NO:81, 82, 85, 86, 88, 89, and 90. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:81. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:82. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:85. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:86. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:88. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:89. In some embodiments, the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:90. 2. Spacer

[0235] In some embodiments, the recombinant receptor (such as the CAR comprising an extracellular 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 form thereof. In some embodiments, at least a portion of the immunoglobulin constant region comprises a hinge region, such as an Ig4 hinge region, and / or C H 1, C H 2 or C H 3 and / or an Fc region. In some embodiments, the constant region or portion is the constant region or portion of human IgG (such as IgG4 or IgG1). In some aspects, the portion of the constant region serves as a spacer region between the antigen-binding domain or a portion thereof (such as the VH and VL of the GPRC5D-binding domain or BCMA-binding 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-expressing 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 suitable for T cell activation or adjusted to optimize the performance of CAR T cells.

[0236] In some embodiments, the spacer has a length that provides increased cellular reactivity after antigen binding compared to the situation in the absence of a spacer or compared to alternative spacers of different lengths (e.g., shorter lengths). In some instances, the length of the spacer is or is about 12 amino acids or its length does not exceed 12 amino acids. In some embodiments, the length of the spacer is at least 100 amino acids, such as a length of at least 110, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 amino acids. 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 and including any integer between the endpoints of any of the listed ranges. In some embodiments, the length of the spacer region is at least about 12 amino acids, 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 length of the spacer is 125 to 300 amino acids, 125 to 250 amino acids, 125 to 230 amino acids, 125 to 200 amino acids, 125 to 180 amino acids, 125 to 150 amino acids, 150 to 300 amino acids, 150 to 250 amino acids, 150 to 230 amino acids, 150 to 200 amino acids, 150 to 180 amino acids, 180 to 300 amino acids, 180 to 250 amino acids, 180 to 230 amino acids, 180 to 200 amino acids, 200 to 300 amino acids, 200 to 250 amino acids, 200 to 230 amino acids, 230 to 300 amino acids, 230 to 250 amino acids, or 250 to 300 amino acids. In some embodiments, the length of the spacer is at least or at least about or is about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, or 229 amino acids, or its length is between any two of the foregoing values.

[0238] Exemplary spacers include the individual IgG hinge, an IgG hinge linked to one or more of the C H 2 and C H 3 domains, or a hinge linked to CH The IgG hinge of the 3 domains. In some embodiments, the IgG hinge, C H 2 and / or C H 3 may be wholly or partly derived from IgG4 or IgG2, such as all or part of human IgG4 or human IgG2. In some embodiments, the spacer may be a chimeric polypeptide containing one or more of the hinge, C H 2 and / or C H 3 sequences from IgG4, IgG2, and / or IgG2 and IgG4. In some embodiments, the hinge region comprises all or part of an IgG4 hinge region and / or an IgG2 hinge region, wherein the IgG4 hinge region is optionally a human IgG4 hinge region and the IgG2 hinge region is optionally a human IgG2 hinge region; C H 2 region contains the IgG4 C H 2 region and / or the IgG2 C H 2 region, wherein the IgG4 C H 2 region is optionally a human IgG4 C H 2 region and the IgG2 C H 2 region is optionally a human IgG2 C H 2 region; and / or C H 3 region contains the IgG4 C H 3 region and / or the IgG2 C H 3 region, wherein the IgG4 C H 3 region is optionally a human IgG4 C H 3 region and the IgG2C H 3 region is optionally a human IgG2 C H 3 region. In some embodiments, the hinge, C H 2 and C H 3 contain all or part of the hinge region, C H 2 and C H 3 from IgG4 respectively. In some embodiments, the hinge region is chimeric and contains the hinge regions from human IgG4 and human IgG2; C H 2 region is chimeric and contains the C H 2 regions from human IgG4 and human IgG2; and / or C H 3 region is chimeric and contains the C H 3 regions from human IgG4 and human IgG2. In some embodiments, the spacer contains an IgG4 / 2 chimeric hinge or a modified IgG4 hinge containing at least one amino acid substitution compared to the human IgG4 hinge region; a human IgG2 / 4 chimeric C H 2 region; and a human IgG4 C H 3 region.

[0239] In some embodiments, the spacer may be wholly or partially derived from IgG4 and / or IgG2 and may contain mutations, such as one or more single amino acid mutations in one or more domains. In some instances, 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 the substitution of asparagine (N) with glutamine (Q) to reduce glycosylation heterogeneity, such as the full-length IgG4 Fc sequence C shown in SEQ ID NO:75 H The N177Q mutation at position 177 in region C2, or the full-length IgG2 Fc sequence C shown in SEQ ID NO:76 H The N176Q mutation at position 176 in region C2. In some embodiments, the spacer is or comprises an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric C H region 2; and an IgG4 C H region 3. In some embodiments, the spacer has a length of about 228 amino acids. In some embodiments, the spacer is as shown in SEQ ID NO:27. In some embodiments, the spacer comprises the amino acid sequence ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(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 eliminate splice sites, such as cryptic splice sites. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:49. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:50. In some embodiments, the coding sequence for the spacer comprises the nucleic acid sequence set forth in SEQ ID NO:73. In some embodiments, the coding sequence for 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 nucleotide sequence of the spacer is optimized to reduce RNA heterogeneity upon expression. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce cryptic splice sites or to reduce the likelihood of a splicing event occurring at the splice site.

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

[0244] In some embodiments, the spacer is encoded by a polynucleotide that has optionally been 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 and / or acceptor sites are present 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, and after expression in a cell, one or more cryptic splice donor and / or acceptor sites in the polynucleotide are eliminated and / or modified to reduce the heterogeneity of the RNA (such as mRNA) transcribed from the construct. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO:49. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO:50. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO:73. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO:74. 3. Transmembrane domain and intracellular signaling components

[0245] The extracellular antigen-binding domain (i.e., the GPRC5D and BCMA-binding domains) is typically linked to one or more intracellular signaling components, such as signaling components that mimic activation achieved via an antigen receptor complex (e.g., in the case of a CAR, the TCR complex) and / or signal via another cell surface receptor. Thus, in some embodiments, a GPRC5D-binding molecule (e.g., an antibody or an antigen-binding fragment thereof) or a BCMA-binding molecule (e.g., an antibody or an antigen-binding fragment thereof) is linked to one or more transmembrane domains (such as the transmembrane domains described herein) and an intracellular signaling domain that contains one or more intracellular components (such as the components described herein). In some embodiments, the VH or VL closest to the cell membrane in the binding domain is linked to the transmembrane domain. Typically, the binding domain or a component thereof (e.g., the VH region or VL region 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 some embodiments, a transmembrane domain that is naturally associated with a domain of a receptor, such as a CAR, is used. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid such domains binding to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0246] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. In the case of a natural source, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. Transmembrane domains include transmembrane domains (i.e., at least containing the following) derived from the α, β, or ζ chains of the T cell receptor, CD3ε, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and / or CD154. For example, in some embodiments, the transmembrane domain can be the CD28 transmembrane domain containing the amino acid sequence shown in SEQ ID NO:18, which can be encoded by the nucleic acid sequence shown in SEQ ID NO:55 or SEQ ID NO:56. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, the synthetic transmembrane domain mainly contains hydrophobic residues, such as leucine and valine. In some aspects, a triplet consisting of phenylalanine, tryptophan, and valine can be found at both ends of the synthetic transmembrane domain. In some embodiments, the linkage is carried out via a linker, spacer, and / or transmembrane domain.

[0247] The intracellular signaling domain has a domain that mimics or approximates signaling via a natural antigen receptor, signaling via a combination of such a receptor and a co-stimulatory receptor, and / or signaling via a co-stimulatory receptor alone. In some embodiments, there is a short oligonucleotide or polypeptide linker, such as a linker between 2 and 10 amino acids in length, such as a linker containing glycine and serine, such as a glycine-serine dyad, that forms a connection between the transmembrane domain of the CAR and the intracellular signaling domain.

[0248] A receptor, such as a CAR, typically includes an intracellular signaling region that includes at least one intracellular signaling component. In some embodiments, the receptor includes an intracellular component or signaling domain of the TCR complex, such as the TCR CD3 chains that mediate T cell activation and cytotoxicity, such as the CD3zeta (CD3-ζ) chain. Thus, in some aspects, a GPRC5D or BCMA binding antibody is linked to one or more cell signaling modules. In some embodiments, the cell signaling module includes a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, such as a CAR, further includes a portion of one or more additional molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR includes a chimeric molecule between CD3-zeta (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0249] In some embodiments, upon CAR engagement, the cytoplasmic domain or intracellular signaling domain of the CAR stimulates and / or activates an immune cell, such as at least one of the normal effector functions or responses of a T cell engineered to express the CAR. For example, in some instances, the CAR induces functions of the T cell, such as cytolytic activity; or T helper cell activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signaling domain of an antigen receptor component or co-stimulatory molecule is used to replace a full-length immune-stimulatory chain, such as when the portion transduces an effector function signal. In some embodiments, one or more intracellular signaling domains include the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects, also include the cytoplasmic sequence of a co-receptor that cooperates with such a receptor in the native environment to initiate signaling after antigen receptor engagement, and / or any derivative or variant of such a molecule, and / or any synthetic sequence having the same functional capacity.

[0250] In the case of a native TCR, full activation typically requires not only signaling through the TCR but also co-stimulatory signals. Thus, in some embodiments, elements for generating secondary or co-stimulatory signals are also included in the CAR to facilitate full activation. In other embodiments, the CAR does not include components for generating co-stimulatory signals. In some aspects, another CAR is expressed in the same cell and provides components for generating secondary or co-stimulatory signals.

[0251] T cell activation is described in some aspects as being mediated by two classes of cytoplasmic signaling sequences: those cytoplasmic signaling sequences that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those cytoplasmic signaling sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such cytoplasmic signaling sequences.

[0252] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary stimulation and / or activation of the TCR complex. A primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing an ITAM include primary cytoplasmic signaling sequences derived from: TCR or CD3ζ, FcRγ, CD3γ, CD3δ, and CD3ε. In some embodiments, the intracellular signaling region in the CAR contains the cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ. In some embodiments, the CD3ζ contains the amino acid sequence shown in SEQ ID NO: 30. In some embodiments, the CD3ζ is encoded by the nucleic acid sequence shown in SEQ ID NO: 55 or SEQ ID NO: 56.

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

[0254] In some embodiments, these stimulatory or activating components are included within one CAR, while the co-stimulatory element is provided by another CAR that recognizes a different antigen. In some embodiments, the CARs include an activating or stimulatory CAR and a co-stimulatory CAR expressed on the same cell (see WO 2014 / 055668). In some aspects, the CAR targeting GPRC5D is the stimulatory or activating CAR; in other aspects, it is the co-stimulatory CAR. In some embodiments, the cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Med., 5(215) (December 2013)), such as a CAR that recognizes a non-GPRC5D antigen, whereby the stimulatory or activating signal delivered via the CAR targeting GPRC5D is attenuated or inhibited upon binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[0255] In certain embodiments, the intracellular signaling domain comprises the CD28 transmembrane and signaling domain linked to the intracellular domain of CD3 (e.g., CD3-ζ). In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and 4-1BB (CD137; TNFRSF9) co-stimulatory domain linked to the CD3ζ intracellular domain.

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

[0257] In some embodiments, the provided embodiments of the anti-GPRC5D CAR comprise an extracellular antigen-binding domain that comprises any anti-GPRC5D antibody or antigen-binding fragment described herein, such as in Section I.1a; 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 a spacer of approximately 228 amino acids in length, or the spacer shown in SEQ ID NO:27, such as a spacer encoded by any of the nucleotide sequences in SEQ ID NOs: 49, 50, 73, and 74; a transmembrane domain, such as the transmembrane domain from human CD28; and an intracellular signaling region that comprises the cytoplasmic signaling domain of the CD3-zeta (CD3ζ) chain and the intracellular signaling domain of a T cell co-stimulatory molecule. Polynucleotides encoding such chimeric antigen receptors are also provided. In some embodiments, the transmembrane domain is or comprises the sequence shown in SEQ ID NO:28. In some embodiments, the intracellular signaling domain of the T cell co-stimulatory 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 shown in SEQ ID NO:29. In some embodiments, the cytoplasmic signaling domain is the human CD3-ζ cytoplasmic signaling domain, such as that shown in SEQ ID NO:30. In some embodiments, the intracellular signaling domain comprises the sequences shown in SEQ ID NO:30 and SEQ ID NO:29. 4. Exemplary CAR

[0258] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:19, the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:17, and the VL region of the BCMA-binding domain.

[0259] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:24, the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:17, and the VL region of the BCMA-binding domain.

[0260] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:21, the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:17, and the VL region of the BCMA-binding domain.

[0261] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:24, the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:17, and the VH region of the BCMA-binding domain.

[0262] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:19, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:19, and the VL region of the BCMA-binding domain.

[0263] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:19, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:24, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:19, and the VH region of the BCMA-binding domain.

[0264] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:21, the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:17, the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:21, and the VL region of the GPRC5D-binding domain.

[0265] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:21, the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:17, the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:21, and the VH region of the GPRC5D-binding domain.

[0266] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:21, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:21, and the VH region of the BCMA-binding domain.

[0267] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:21, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:21, and the VH region of the BCMA-binding domain.

[0268] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:22, the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:17, the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:22, and the VL region of the GPRC5D-binding domain.

[0269] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VH region of the BCMA-binding domain, the linker shown in SEQ ID NO:22, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:22, and the VL region of the BCMA-binding domain.

[0270] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:22, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:17, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:22, and the VH region of the BCMA-binding domain.

[0271] In some embodiments, the CAR comprises an extracellular antigen-binding domain that, in order from the N-terminus to the C-terminus, comprises: the VL region of the BCMA-binding domain, the linker shown in SEQ ID NO:22, the VH region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:24, the VL region of the GPRC5D-binding domain, the linker shown in SEQ ID NO:22, and the VH region of the BCMA-binding domain.

[0272] In some embodiments, the CAR comprises an extracellular antigen-binding domain that comprises the spacer shown in SEQ ID NO:27. In some embodiments, the CAR comprises the transmembrane domain shown in SEQ ID NO:28. In some embodiments, the CAR comprises an intracellular signaling domain that comprises the amino acid sequences shown in SEQ ID NO:29 and 30.

[0273] In some embodiments, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO:31-44, or is encoded by the nucleotide sequence shown in any one of SEQ ID NO:105-120. In some embodiments, the CAR comprises the amino acid sequence shown in any one of SEQ ID NO:31-44. In some embodiments, the CAR is encoded by the nucleotide sequence shown in any one of SEQ ID NO:105-120.

[0274] In some embodiments, the CAR comprises an amino acid 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: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:105. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:31, or is encoded by the nucleotide sequence shown in SEQ ID NO:105. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:105. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:31. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells, such as cancer cells, such as plasma cells of a subject with multiple myeloma).

[0275] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:32, or is encoded by the nucleotide sequence shown in SEQ ID NO:106. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:32. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:106. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells, such as cancer cells, such as plasma cells of a subject with multiple myeloma).

[0276] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:33, or is encoded by the nucleotide sequence shown in SEQ ID NO:107. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:33. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:107. In some 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, such as cancer cells, such as plasma cells of a subject with multiple myeloma).

[0277] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:34, or is encoded by the nucleotide sequence shown in SEQ ID NO:108. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:34. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:108. In some 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, such as cancer cells, such as plasma cells of a subject with multiple myeloma).

[0278] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:35, or is encoded by the nucleotide sequence shown in SEQ ID NO:109. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:35. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:109. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells, such as cancer cells, such as plasma cells of a subject with multiple myeloma).

[0279] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:36, or is encoded by the nucleotide sequence shown in SEQ ID NO:110. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:36. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:110. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells, such as cancer cells, such as plasma cells of a subject with multiple myeloma).

[0280] In some embodiments, the CAR comprises an amino acid 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: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 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:119. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:37, or is encoded by the nucleotide sequence shown in SEQ ID NO:111. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:37, or is encoded by the nucleotide sequence shown in SEQ ID NO:119. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:37. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ IDNO:111. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:119. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells (such as cancer cells, such as plasma cells of a subject with multiple myeloma)).

[0281] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:38, or is encoded by the nucleotide sequence shown in SEQ ID NO:112. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:38. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:112. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells (such as cancer cells, such as plasma cells of a subject with multiple myeloma)).

[0282] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:39, or is encoded by the nucleotide sequence shown in SEQ ID NO:113. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:39. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:113. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:112. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells (such as cancer cells, such as plasma cells of a subject with multiple myeloma)).

[0283] In some embodiments, the CAR comprises an amino acid 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: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 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:120. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:40, or is encoded by the nucleotide sequence shown in SEQID NO:114. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:40, or is encoded by the nucleotide sequence shown in SEQ ID NO:120. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:40. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:114. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:120. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells (such as cancer cells, such as plasma cells of a subject with multiple myeloma)).

[0284] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:41, or is encoded by the nucleotide sequence shown in SEQ ID NO:115. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:41. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:115. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:120. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells (such as cancer cells, such as plasma cells of a subject with multiple myeloma)).

[0285] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:42, or is encoded by the nucleotide sequence shown in SEQ ID NO:116. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:42. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:116. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells (such as cancer cells, such as plasma cells of a subject with multiple myeloma)).

[0286] In some embodiments, the CAR comprises an amino acid 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: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 shown in SEQ ID NO:43, or is encoded by the nucleotide sequence shown in SEQ ID NO:117. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:43. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:117. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells, such as cancer cells, such as plasma cells of a subject 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 shown in SEQ ID NO:44, or is encoded by the nucleotide sequence shown in SEQ ID NO:118. In some embodiments, the CAR comprises the amino acid sequence shown in SEQ ID NO:44. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:118. In some such embodiments, the CAR is a dual-targeting CAR that directly binds to GPRC5D and BCMA (such as expressed on the surface of cells, such as cancer cells, such as plasma cells of a subject with multiple myeloma). 5. Exemplary Features

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

[0289] In some embodiments, the antibody specifically binds to the human GPRC5D protein, such as an epitope or region of the human GPRC5D protein, such as the 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, as measured, for example, by radioimmunoassay (RIA), the degree to which the anti-GPRC5D antibody or antigen-binding domain or CAR binds to an unrelated non-GPRC5D protein, such as a non-human GPRC5D protein or other non-GPRC5D protein, is less than or less than about 10% of the binding of the antibody or antigen-binding domain or CAR to the human GPRC5D protein or human membrane-bound GPRC5D. In some embodiments, there are antibody or antigen-binding domains or CARs in the provided CARs in which the binding to murine GPRC5D protein is less than or is or is about 10% of the binding of the antibody to human GPRC5D protein. In some embodiments, the antibody or antigen-binding domain in the provided CARs is an antibody in which the binding to cynomolgus macaque GPRC5D protein is less than or is or is about 10% of the binding of the antibody to human GPRC5D protein. In some embodiments, the antibody or antigen-binding domain in the provided CARs is an antibody in which the binding to cynomolgus macaque GPRC5D protein and / or murine GPRC5D protein is similar to or substantially equivalent to the binding of the antibody to human GPRC5D protein.

[0291] In some embodiments, the antibody in the provided CAR is capable of binding to a GPRC5D protein, such as a human GPRC5D protein, with at least a certain affinity, which is measured by any of a variety of known methods. In some embodiments, the affinity is represented by the equilibrium dissociation constant (K D ); in some embodiments, the affinity is represented by EC 50 .

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

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

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

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

[0296] In some embodiments, the antibody in the provided CAR is capable of binding to the BCMA protein, such as the human BCMA protein, with at least a certain affinity, which is measured by any of a variety of known methods. In some embodiments, the affinity is represented by the equilibrium dissociation constant (K D ); in some embodiments, the affinity is represented by EC 50 .

[0297] A variety of assays are known for assessing binding affinity and / or determining whether a binding molecule (e.g., an antibody or a fragment thereof) specifically binds to a particular ligand (e.g., an antigen, such as the BCMA protein). Determining the binding affinity of a binding molecule, such as an antibody, for an antigen, such as BCMA, such as human BCMA or cynomolgus macaque BCMA or mouse BCMA, is within the skill of the art, such as by using any of a variety of binding assays well known in the art. For example, in some embodiments, An instrument that uses surface plasmon resonance (SPR) analysis to determine the binding kinetics and constants of complexes between two proteins (e.g., an antibody or its fragment and an antigen, such as the BCMA protein) (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or equivalents).

[0298] SPR measures the change in the concentration of molecules at the surface when molecules bind to or dissociate from the sensor surface. The change in the SPR signal is proportional to the change in the mass concentration near the surface, thereby allowing the 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 buffer passes over the chip. Other suitable assays for measuring the binding of one protein to another include, for example, immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), or by monitoring changes in the spectral or optical properties of the protein by fluorescence, UV absorption, circular dichroism, or nuclear magnetic resonance (NMR). Other exemplary assays include (but are not limited to) Western blot, ELISA, analytical ultracentrifugation, spectroscopy, flow cytometry, sequencing, and other methods for monitoring the binding of expressed polynucleotides or proteins.

[0299] In some embodiments, the binding molecule, such as an antibody or its fragment or antigen-binding domain of a CAR, binds (e.g., specifically binds) to an antigen, such as the GPRC5D protein or an epitope thereof, with an affinity or K 5 M -1 equal to or greater than 10 A (i.e., the equilibrium association constant of a specific binding interaction, with units of 1 / M; in the case of a bimolecular interaction, equal to the ratio of the association rate [k on or k a to the dissociation rate [k off or k d ). In some embodiments, the K D (i.e., the equilibrium dissociation constant of a specific binding interaction, with units of M; in the case of a bimolecular interaction, equal to the ratio of the dissociation rate [k off or k d to the association rate [k on or k a ) of the binding affinity exhibited by the antibody or its fragment or antigen-binding domain of the CAR for a peptide epitope is equal to or less than 10-5 M. For example, the equilibrium dissociation constant K D is between 10 -5 M and 10 -13 M, such as between 10 -7 M and 10 -11 M, between 10 -8 M and 10 -10 M, or between 10 -9 M and 10 -10 M. The association rate (association rate constant; k on or k a ; in units of 1 / Ms) and the dissociation rate (dissociation rate constant; k off or k d ; in units of 1 / s) can be determined using any of the analytical methods known in the art, such as surface plasmon resonance (SPR).

[0300] In some embodiments, the binding affinity (EC 50 ) and / or dissociation constant of an antibody (e.g., an antigen-binding fragment) or antigen-binding domain of a CAR to a GPRC5D protein, such as a human GPRC5D protein, is from or about 0.01 nM to about 500 nM, from or about 0.01 nM to about 400 nM, from or about 0.01 nM to about 100 nM, from or about 0.01 nM to about 50 nM, from or about 0.01 nM to about 10 nM, from or about 0.01 nM to about 1 nM, from or about 0.01 nM to about 0.1 nM, from or about 0.1 nM to about 500 nM, from or about 0.1 nM to about 400 nM, from or about 0.1 nM to about 100 nM, from or about 0.1 nM to about 50 nM, from or about 0.1 nM to about 10 nM, from or about 0.1 nM to about 1 nM, from or about 0.5 nM to about 200 nM, from or about 1 nM to about 500 nM, from or about 1 nM to about 100 nM, from or about 1 nM to about 50 nM, from or about 1 nM to about 10 nM, from or about 2 nM to about 50 nM, from or about 10 nM to about 500 nM, from or about 10 nM to about 100 nM, from or about 10 nM to about 50 nM, from or about 50 nM to about 500 nM, from or about 50 nM to about 100 nM, or from or about 100 nM to about 500 nM. In certain embodiments, the binding affinity (EC 50 ) and / or equilibrium dissociation constant K Dis for 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 lower. In some embodiments, the antibody binds to the GPRC5D protein, such as the human GPRC5D protein, with a sub-nanomolar binding affinity, e.g., with a binding affinity of less than about 1 nM, such as less than 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 lower.

[0301] In some embodiments, the binding affinity can be classified as high affinity or low affinity. In some cases, the binding molecule (e.g., an antibody or a fragment thereof) or antigen-binding domain of a CAR that exhibits low to medium affinity binding exhibits a K A of up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 . In some cases, the binding molecule (e.g., an antibody or a fragment thereof) that exhibits high affinity binding to a specific epitope interacts with such an epitope with a K A 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 . In some embodiments, the binding affinity (EC 50 ) and / or the equilibrium dissociation constant K Dis from or 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 of the binding molecule, such as an anti-GPRC5D antibody or a fragment or antigen-binding domain thereof of a CAR, to the GPRC5D protein is or is about or less than 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 lower. 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 antigen-binding domain of a CAR, for different antigens, such as 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 constants K D for different antigens, such as GPRC5D proteins from different species, such as from human, cynomolgus macaque or mouse, 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, such as the anti-GPRC5D antibody binds to human GPRC5D and species variant GPRC5D to a similar extent, such as the ratio of K D for human GPRC5D to K D for the species variant GPRC5D is or is about 1. In some embodiments, the species cross-reactivity of an anti-GPRC5D antibody or antigen-binding domain of a CAR can be low, such as the anti-GPRC5D antibody has a high affinity for human GPRC5D but a low affinity for the species variant GPRC5D, or vice versa. For example, the ratio of K D for the species variant GPRC5D to K DThe ratio exceeds 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or higher, and the anti-GPRC5D antibody has low species cross-reactivity. The degree of species cross-reactivity can be compared with the species cross-reactivity of a known antibody, such as a reference antibody.

[0303] The provided bispecific CARs include CARs that exhibit antigen-dependent activity or signaling, i.e., there is significantly no signaling activity or there is background level signaling activity in the absence of an antigen, such as GPRC5D. Thus, in some aspects, the provided CARs do not exhibit or exhibit no more than background level, or exhibit permissive or low levels of constitutive signaling or antigen-independent activity or signaling in the absence of an antigen, such as GPRC5D. In some embodiments, the provided cells expressing the bispecific CAR exhibit biological activity or function, including cytotoxic activity, cytokine production, and proliferative capacity.

[0304] In some embodiments, the binding molecule, such as an antibody or a fragment or antigen-binding domain thereof of the CAR, binds (e.g., specifically binds) to an antigen, such as the BCMA protein (e.g., SEQ ID NO: 60) or an epitope therein, with an affinity equal to or greater than 10 5 M -1 or K A (i.e., the equilibrium association constant of a specific binding interaction, with units of 1 / M; in the case of a presumed bimolecular interaction, equal to the association rate [k on or k a of this association reaction to the dissociation rate [k off or k d ratio). In some embodiments, the K D (i.e., the equilibrium dissociation constant of a specific binding interaction, with units of M; in the case of a presumed bimolecular interaction, equal to the dissociation rate [k off or k d of this association reaction to the association rate [k on or k a ratio) of the binding affinity exhibited by the antibody or a fragment or antigen-binding domain thereof of the CAR for a peptide epitope is equal to or less than 10 -5 M. For example, the equilibrium dissociation constant K D ranges from 10 -5 M to 10 -13 M, such as 10 -7 M to 10 -11 M, 10 -8 M to 10 -10 M or 10 -9 M to 10 -10M. The association rate (association rate constant; k on or k a ; in units of 1 / Ms) and the dissociation rate (dissociation rate constant; k off or k d ; in units of 1 / s) can be determined using any of the assay methods known in the art (e.g., surface plasmon resonance (SPR)).

[0305] In some embodiments, the binding affinity (EC 50 ) and / or dissociation constant of an antibody (e.g., antigen-binding fragment) or antigen-binding domain of a CAR with a BCMA protein (such as a human BCMA protein) is from or about 0.01 nM to about 500 nM, from or about 0.01 nM to about 400 nM, from or about 0.01 nM to about 100 nM, from or about 0.01 nM to about 50 nM, from or about 0.01 nM to about 10 nM, from or about 0.01 nM to about 1 nM, from or about 0.01 nM to about 0.1 nM, from or about 0.1 nM to about 500 nM, from or about 0.1 nM to about 400 nM, from or about 0.1 nM to about 100 nM, from or about 0.1 nM to about 50 nM, from or about 0.1 nM to about 10 nM, from or about 0.1 nM to about 1 nM, from or about 0.5 nM to about 200 nM, from or about 1 nM to about 500 nM, from or about 1 nM to about 100 nM, from or about 1 nM to about 50 nM, from or about 1 nM to about 10 nM, from or about 2 nM to about 50 nM, from or about 10 nM to about 500 nM, from or about 10 nM to about 100 nM, from or about 10 nM to about 50 nM, from or about 50 nM to about 500 nM, from or about 50 nM to about 100 nM, or from or about 100 nM to about 500 nM. In certain embodiments, the binding affinity (EC 50 ) and / or equilibrium dissociation constant K Dis for 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 lower. In some embodiments, the antibody binds to the BCMA protein, such as the human BCMA protein, with a sub-nanomolar binding affinity, e.g., with a binding affinity of less than about 1 nM, such as less than 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 lower.

[0306] In some embodiments, the binding affinity can be classified as high affinity or low affinity. In some cases, a binding molecule (e.g., an antibody or a fragment thereof) or an antigen-binding domain of a CAR that exhibits low to medium affinity binding exhibits a K A of at most 10 7 M -1 , at most 10 6 M -1 , at most 10 5 M -1 . In some cases, a binding molecule (e.g., an antibody or a fragment thereof) that exhibits high affinity binding to a specific epitope interacts with such an epitope with a K A 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 . In some embodiments, the binding affinity (EC 50 ) and / or the equilibrium dissociation constant K Dis from or 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 of the binding molecule (e.g., an anti-BCMA antibody or a fragment or antigen-binding domain thereof of a CAR) to the BCMA protein is or is 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 lower. 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 a binding molecule (such as an anti-BCMA antibody or antigen-binding domain of a CAR) 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 D for different antigens (e.g., BCMA proteins from different species, such as from human, cynomolgus macaque, or mouse) can be compared to determine species cross-reactivity. In some embodiments, the species cross-reactivity of an anti-BCMA antibody or antigen-binding domain of a CAR can be high, e.g., the anti-BCMA antibody binds to human BCMA and species variant BCMA to a similar extent, e.g., the ratio of K D for human BCMA to K D for the species variant BCMA is or is about 1. In some embodiments, the species cross-reactivity of an anti-BCMA antibody or antigen-binding domain of a CAR can be low, e.g., the anti-BCMA antibody has high affinity for human BCMA but low affinity for the species variant BCMA, or vice versa. For example, the ratio of K D for the species variant BCMA to K D for human BCMA exceeds 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000, or higher, and the anti-BCMA 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.

[0308] The provided CARs include those that exhibit antigen-dependent activity or signaling, i.e., CARs that exhibit significantly absent or background levels of signaling activity in the absence of antigen (e.g., BCMA). Thus, in some aspects, the provided CARs do not exhibit or exhibit no more than background levels, or exhibit permissive or low levels of tonic signaling or antigen-independent activity or signaling, in the absence of antigen (e.g., BCMA). In some embodiments, the provided cells expressing bispecific CARs exhibit biological activity or function, including cytotoxic activity, cytokine production, and proliferative capacity.

[0309] In some embodiments, the biological or functional activity of the chimeric receptor, such as cytotoxic activity, can be measured using any one of a variety of known methods. This activity can be evaluated or assayed in vitro or in vivo. In some embodiments, the activity can be evaluated after administering the cells to a subject (e.g., a human). Parameters for evaluation include the specific binding of genetically engineered or native T cells or other immune cells to an antigen, e.g., in vivo, such as by imaging assays; or ex vivo, such as by ELISA or flow cytometry assays. In certain embodiments, the ability of the engineered cells to lyse target cells can be measured using any suitable method known in the art, such as the cytotoxicity assays described in, for example, 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 the cells can also be measured by analyzing the expression and / or secretion of certain cytokines, such as interleukin-2 (IL-2), interferon-γ (IFNγ), interleukin-4 (IL-4), TNF-α (TNFα), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD107a, and / or TGF-β (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, where the reactivity (e.g., proliferation) of the cells in response to the relevant cytokine is tested in the presence of the test sample. In some aspects, the biological activity is measured by evaluating clinical outcomes, such as a decrease in tumor burden or load.

[0310] In some aspects, monitoring antigen-independent activity of a reporter somatic cell line and / or obligatory signaling via cells expressing a bispecific CAR can be employed. In some embodiments, a T cell line (such as Jurkat cells, which are BCMA-negative / GPRC5D-negative) contains a reporter molecule, such as a fluorescent protein or other detectable molecule, such as a red fluorescent protein, expressed under the control of an endogenous Nur77 transcriptional regulatory element. In some embodiments, Nur77 reporter expression is cell-intrinsic and depends on signaling via a recombinant reporter containing a primary activation signal in T cells, a signaling domain consisting of a T cell receptor (TCR), and / or a signaling domain containing an immunoreceptor tyrosine-based activation motif (ITAM) (such as the CD3ζ chain). Nur77 expression is generally not affected by other signaling pathways such as cytokine signaling or Toll-like receptor (TLR) signaling, which can act in an extracellular manner and may not depend on signaling via a recombinant receptor. Thus, cells expressing only an exogenous recombinant receptor (such as a bispecific CAR) containing an appropriate signaling region are capable of expressing Nur77 upon stimulation (such as binding to a specific antigen). In some cases, Nur77 expression can also exhibit a dose-dependent response to the amount of stimulation (such as antigen).

[0311] In some embodiments, the provided bispecific CAR exhibits improved expression on the cell surface compared to an alternative CAR having the same amino acid sequence but encoded by a nucleotide sequence that lacks splice sites and / or is not codon-optimized. In some embodiments, the expression of the recombinant receptor on the cell surface can be evaluated. Methods for determining the expression of a recombinant receptor on the cell surface can include using a chimeric antigen receptor (CAR)-specific antibody (such as Brentjens et al., Sci. Transl. Med. March 2013; 5(177):177ra38), protein L (Zheng et al., J. Transl. Med. February 2012; 10:29), an epitope tag, and a monoclonal antibody that specifically binds to the CAR polypeptide (see International Patent Application Publication No. WO2014190273). In some embodiments, the expression of the recombinant receptor on the surface of a cell (such as a primary T cell) can be evaluated, for example, by flow cytometry using a binding molecule that can detectably bind to the recombinant receptor or a portion thereof. In some embodiments, the binding molecule for detecting the expression of the recombinant receptor is or comprises an anti-idiotypic antibody, such as an anti-idiotypic agonistic antibody or a portion thereof that is specific for a binding domain (such as an scFv). In some embodiments, the binding molecule is or comprises an isolated or purified antigen, such as an antigen expressed recombinantly. II. Polynucleotides Encoding One or More Recombinant Receptors

[0312] Also provided are polynucleotides encoding chimeric antigen receptors and / or portions thereof (e.g., chains). The provided polynucleotides include polynucleotides encoding bispecific chimeric antigen receptors (e.g., antigen-binding fragments) that bind BCMA and GPRC5D as described herein. The polynucleotides can include polynucleotides encompassing natural and / or non-naturally occurring nucleotides and bases, such as polynucleotides including backbone modifications. The terms "nucleic acid molecule", "nucleic acid", and "polynucleotide" are used interchangeably and refer to nucleotide polymers. Such nucleotide polymers can contain natural and / or non-natural nucleotides and include (but are not limited to) DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides comprising a nucleic acid molecule or polynucleotide.

[0313] In some embodiments, the extracellular binding domain comprises, from the amino-terminus to the carboxy-terminus: 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; one of the VH and VL regions of the BCMA binding domain; and the other of the VH and VL regions of the BCMA binding domain. In some embodiments, the extracellular binding domain comprises, from the amino-terminus to the carboxy-terminus: 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. In some cases, the polynucleotide encoding the GPRC5D binding domain comprises a signal sequence encoding a signal peptide, in some cases upstream of the nucleic acid sequence encoding the GPRC5D binding domain, or ligated to 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 comprises a signal sequence encoding a signal peptide. In some aspects, the signal sequence can encode a signal peptide from a native polypeptide. In other aspects, the signal sequence can encode a heterologous or non-native signal peptide. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the IgGκ chain shown in SEQ ID NO:92 or a signal peptide encoded by the nucleotide sequence shown in SEQ ID NO:91 or 93-96. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the GMCSFRα chain shown in SEQ ID NO:98 or a signal peptide encoded by the nucleotide sequence shown in SEQ ID NO:97. In some aspects, non-limiting exemplary signal peptides comprise the signal peptide of the CD8α signal peptide shown in SEQ ID NO:99. In some aspects, non-limiting exemplary signal peptides comprise the signal peptide of the CD33 signal peptide shown in SEQ ID NO:72. In some cases, the polynucleotide encoding the GPRC5D binding receptor can contain a nucleic acid sequence encoding an additional molecule (such as an alternative marker or other marker), or can contain additional components such as a promoter, regulatory element, and / or polycistronic element. In some embodiments, the nucleic acid sequence encoding the GPRC5D binding domain can be operably linked to any of these additional components.

[0314] In some embodiments, the extracellular binding domain comprises, from the amino terminus to the carboxy terminus: 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; one of the VH and VL regions of the GPRC5D binding domain; and the other of the VH and VL regions of the GPRC5D binding domain. In some embodiments, the extracellular binding domain comprises, from the amino terminus to the carboxy terminus: 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. In some cases, the polynucleotide encoding the BCMA binding domain contains a signal sequence encoding a signal peptide, in some cases upstream of the nucleic acid sequence encoding the BCMA binding domain or linked to 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 aspects, the signal sequence may encode a signal peptide from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the IgGκ chain shown in SEQ ID NO:92 or the signal peptide encoded by the nucleotide sequence shown in SEQ ID NO:271 or 93 - 96. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the GMCSFRα chain shown in SEQ ID NO:98 or the signal peptide encoded by the nucleotide sequence shown in SEQ ID NO:97. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the CD8α signal peptide shown in SEQ ID NO:99. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the CD33 signal peptide shown in SEQ ID NO:72. In some cases, the polynucleotide encoding the BCMA binding receptor may contain a nucleic acid sequence encoding an additional molecule (such as an alternative marker or other marker), or may contain additional components such as a promoter, regulatory element, and / or polycistronic element. In some embodiments, the nucleic acid sequence encoding the BCMA binding domain may be operably linked to any of these additional components.

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

[0316] In some embodiments, the CARs provided by the present application include CARs encoded by polynucleotides that are optimized or contain certain features designed for optimization (such as codon usage optimization) to reduce RNA heterogeneity and / or modification, such as increasing the expression of the encoded receptor (such as surface expression) or making the expression of the encoded receptor (such as surface expression) more consistent among multiple cell product batches. In some embodiments, the polynucleotides encoding the GPRC5D binding domain and the BCMA binding domain are modified, such as to remove cryptic or hidden splice sites, compared to a reference polynucleotide, to reduce RNA heterogeneity. In some embodiments, the polynucleotides encoding the GPRC5D binding and BCMA binding domains are codon-optimized, such as for expression in mammalian (e.g., human) cells (such as in human T cells). In some aspects, when expressed in cells, the modified polynucleotides result in improved expression (such as surface expression), such as increased expression levels or more uniform or consistent expression. Such polynucleotides can be used in constructs for generating engineered cells expressing the encoded GPRC5D binding and BCMA binding domains. Accordingly, cells expressing the recombinant receptor and their use in adoptive cell therapy (such as for treating diseases and disorders associated with GPRC5D and / or BCMA expression, such as multiple myeloma) are also provided, wherein the recombinant receptor is encoded by the polynucleotides provided herein.

[0317] Also provided are cells engineered to express the polynucleotides provided, such as T cells, the polynucleotides including polynucleotides encoding the GPRC5D binding domain and the 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 in the polynucleotide construct are modified to reduce the heterogeneity of the RNA (such as mRNA) transcribed from the construct after expression in the cells. 1. Codon Optimization

[0318] In some embodiments, the polynucleotide is modified by optimizing codons for expression in humans. In some aspects, codon optimization can be considered before and / or after the steps for splice site identification and / or splice site elimination, and / or at each iterative step for reducing RNA heterogeneity. Codon optimization generally involves balancing the percentage of selected codons with the abundance of human transfer RNAs (e.g., the abundances disclosed), such that there is no overloading or limitation. 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 the transfected or transduced gene or nucleic acid and the host cell can have an impact on protein expression by the nucleic acid molecule. Table 2 below illustrates an exemplary table of human codon usage frequencies. In some embodiments, to generate a codon-optimized nucleic acid sequence, codons are selected to balance the codons with human usage frequencies. The redundancy of amino acid codons allows different codons to encode an amino acid, as depicted in Table 2. When selecting codons for replacement, it is required that the resulting mutation is a silent mutation such that the codon change does not affect the amino acid sequence. Generally, the last nucleotide of the codon (e.g., at the third position) can remain unchanged without affecting the amino acid sequence.

[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). According to the human codon usage frequencies set forth in Table 2 above, the respective usage frequencies of these codons are 15.2, 17.7, 12.2, 4.4, 12.1, and 19.5. Since TCG corresponds to 4.4%, the tRNA for this codon will be limiting when this codon is commonly used in gene synthesis. During the codon optimization process, the goal is to balance the usage of each codon with the normal usage frequencies in the animal species in which the transgene is intended to be expressed. 2. Splice Sites

[0320] A polynucleotide 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 nucleic acid sequence is then synthesized and used to transduce cells to test splicing as indicated by RNA heterogeneity.

[0321] The present disclosure also provides polynucleotides, such as polynucleotides encoding any of the antibodies, receptors (such as antigen receptors, such as chimeric antigen receptors), and / or GPRC5D-specific and / or BCMA-specific binding domains provided herein, which are modified or have been modified to reduce heterogeneity or contain one or more of the nucleic acid sequences observed herein (such as by optimization methods), thereby improving the characteristics of the polypeptides, such as CARs, as compared to polypeptides containing different reference sequences or unmodified polypeptides. Such characteristics include improved RNA heterogeneity, such as heterogeneity caused 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, such as increased levels, uniformity, or consistency of expression in cells engineered to express these polypeptides or different therapeutic cell compositions.

[0322] RNA heterogeneity can be determined relative to the initial sequence by collecting RNA from expressing cells, amplifying using reverse transcriptase polymerase chain reaction (RT-PCR), and resolving using agarose gel electrophoresis, thereby identifying splice sites in the polynucleotide sequence. In some cases, the modified sequence can be resubmitted to a gene synthesis provider for further codon optimization and splice site removal, followed by further cryptic splice site evaluation, modification, synthesis, and testing until the RNA on the agarose gel exhibits minimal RNA heterogeneity.

[0323] Also provided are polynucleotides modified to eliminate splice sites, such as cryptic splice sites. In nature, genomic nucleic acid sequences in mammalian cells generally undergo processing either during or immediately after transcription, where in some cases, nascent precursor messenger ribonucleic acid (pre-mRNA) transcribed from genomic deoxyribonucleic acid (DNA) sequences is edited by splicing to remove introns and then exons are joined in eukaryotic cells. The consensus sequences of splice sites are known, but in some respects, determining the specific nucleotide information of splice sites can be complex and may not be obvious based on available methods. Cryptic splice sites are splice sites that cannot be predicted based on standard consensus sequences and are variably activated. Thus, when expressed in eukaryotic cells, alternative splicing of pre-mRNA at cryptic splice sites results in heterogeneity in the transcribed mRNA product.

[0324] Polynucleotides generated for transgene expression are typically constructed from nucleic acid sequences that do not contain introns, such as complementary DNA (cDNA) or portions thereof. Thus, it is expected that such sequences will not undergo splicing. However, the presence of cryptic splice sites within cDNA sequences can result in unexpected or undesired splicing reactions and heterogeneity in the transcribed mRNA. Such heterogeneity leads to the translation of unexpected protein products, such as truncated protein products with variable amino acid sequences that exhibit regulated expression and / or activity.

[0325] In some embodiments, elimination of splice sites, such as cryptic splice sites, can improve or optimize the expression of transgenic products, such as polypeptides translated from transgenes, such as bispecific CAR polypeptides. Splicing at cryptic splice sites in the encoded transgene, such as a CAR molecule encoding a GPRC5D binding domain and a BCMA binding domain, can reduce protein expression, such as expression on the cell surface, and / or reduce functionality, such as reduce intracellular signaling. Polynucleotides encoding bispecific CAR proteins are provided herein that have been optimized to reduce or eliminate cryptic splice sites. Also provided herein are polynucleotides encoding bispecific CAR proteins that have been optimized for codon expression; and / or in which one or more sequences, such as sequences identified by the methods or observations herein regarding splice sites, are present; and / or in which the identified splice sites, such as any of the splice sites identified herein, are absent. Among the provided polynucleotides are polynucleotides that exhibit a lower degree of RNA heterogeneity or splicing forms when expressed and / or introduced into a specified cell type, such as human T cells, such as primary human T cells, under certain conditions, as well as cells, compositions, and articles containing such polypeptides and / or exhibiting such properties. In some embodiments, the RNA heterogeneity of the transcribed RNA is reduced by more than or by about 10%, 15%, 20%, 25%, 30%, 40%, 50% or higher percentages compared to polynucleotides not modified to remove cryptic splice sites and / or subjected to codon optimization. In some embodiments, the provided polynucleotides encoding bispecific CAR exhibit at least 70%, 75%, 80%, 85%, 90% or 95% or higher percentage of RNA homogeneity of the transcribed RNA.

[0326] RNA heterogeneity can be determined by any one of a variety of methods provided, described, or known herein. In some embodiments, the RNA heterogeneity of a transcribed nucleic acid is determined by amplifying the transcribed nucleic acid using, e.g., reverse transcriptase polymerase chain reaction (RT-PCR), followed by detecting one or more differences of one or more amplification products, such as size differences. In some embodiments, RNA heterogeneity is determined based on the number of amplification products of different sizes or the ratio of the various amplification products of different sizes. In some embodiments, RNA, such as total RNA or cytoplasmic polyadenylated RNA, is collected from cells expressing the transgene to be optimized and amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using primers specific for the 5' untranslated region (5'UTR) (in some cases, which corresponds to a portion of the promoter sequence in the expression vector) upstream of the transgene in the transcribed RNA and primers specific for the 3' untranslated region (3'UTR) downstream of the expressed transgene in the transcribed RNA sequence or primers specific for sequences within the transgene. 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. One of ordinary skill in the art can resolve RNA, such as messenger RNA, and analyze its heterogeneity by several methods. Non-limiting exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical ultracentrifugation, field-flow fractionation, and chromatography, such as size-exclusion chromatography or liquid chromatography.

[0327] In some aspects, the presence of potential cryptic splice sites (splice donor and / or acceptor sites) in transcripts, such as transgene transcripts, can give rise to RNA heterogeneity of the transcripts after expression in cells. In some embodiments, one or more potential splice sites that may be present in a transgene transcript, are undesirable, and / or may be generated by various underlying sequences in the transgene transcript are identified after codon optimization of the transcript and / or by mutation or transcriptional errors or mistakes. In some aspects of the provided embodiments, splice donor sites and splice acceptor sites are identified independently. In some embodiments, the splice acceptor and / or donor sites are canonical, non-canonical, and / or cryptic splice acceptor and / or donor sites.

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

[0329] In some aspects, identifying one or more splice sites in a nucleic acid sequence is an iterative process. In some embodiments, splice sites can be identified using splice site and / or codon optimization prediction tools, such as by submitting an initial or reference sequence encoding a transgene (such as a bispecific CAR or the GPRC5D or BCMA binding domain contained therein) to a database, gene synthesis vendor, or other source capable of computationally or algorithmically comparing the initial or reference sequence to identify or predict splice sites and / or 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 other or additional splice site prediction tools are used to perform one or more other evaluations on the sequence, such as the modified or corrected nucleic acid sequence, to further evaluate splice site removal, such as cryptic splice site removal.

[0330] In some aspects, RNA heterogeneity can be caused by the activity of the spliceosome present in eukaryotic cells. In some aspects, splicing generally occurs in a series of reactions catalyzed by the spliceosome. The consensus sequences of splice sites are known, but in some aspects, determining the specific nucleotide information of splice sites can be complex and may not be obvious based on available methods. Cryptic splice sites are splice sites that cannot be predicted based on the standard consensus sequences and are variably activated. Thus, after expression in eukaryotic cells, alternative splicing of the precursor mRNA at cryptic splice sites results in heterogeneity in the transcribed mRNA products. In some cases, within the intron of the spliceosome, the splicing event requires a donor site (usually at the 5' end of the intron), a branch site (near the 3' end of the intron), and an acceptor site (at the 3' end of the intron). The splice donor site can include the GU sequence at the 5' end of the intron, which has a relatively large region of low conservation. The splice acceptor site at the 3' end of the intron can terminate with the AG sequence.

[0331] In some embodiments, splice sites, including potential cryptic splice sites, can be identified by comparing a sequence to known splice site sequences, such as those in a sequence database. In some embodiments, splice sites can be computationally identified by submitting a nucleotide sequence for analysis using a splice site prediction tool, such as Human Splice Finder (Desmet et al., Nucl. Acids Res. 37(9):e67 (2009)), the 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 identify potential splice sites and the probability of a splicing event at such sites. Additional splicing prediction tools include RegRNA, ESEfinder, and the MIT splicing predictor. Splice site prediction tools, such as GeneSplicer, have been successfully trained and / or tested on databases for different species, such as human, Drosophila melanogaster, Plasmodium falciparum, Arabidopsis thaliana, and rice. In some embodiments, different prediction tools can be adapted for different ranges on different databases and / or for different species. In some embodiments, one or more prediction tools are selected based on their utility in certain databases and / or for certain species. See, e.g., 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 sites. In some embodiments, splice site prediction tools that can be employed can run locally; can be retrained with a set of data at the user's location; can use a database for a particular species, such as human; can be compiled for multiple platforms; allow real-time prediction of sequence selection; and / or are open source software certified by OSI, whereby a particular tool or plug-in can be modified. Exemplary tools that can be employed include NNSplice, GeneSplicer, or both.

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

[0334] In some embodiments, the prediction score of a particular splice site is compared to a threshold score or a reference score to identify or determine a particular splice site as a candidate for elimination or removal. For example, in some embodiments, when the prediction score is greater than or not less than the threshold score or the reference score, the predicted splice site is identified as a potential splice site. In some aspects, considerations regarding the elimination or removal of a particular splice site include the comparison of the prediction score to the reference score or the threshold score; and whether the particular splice site is desired or intended (e.g., when the splicing event is more favorable or when splicing events are required to regulate transcription and / or translation). In some aspects, when determining a particular donor and / or acceptor site to be eliminated or removed, the likelihood that the resulting splice variant will lose a required function or be functionally impaired can also be considered. In some aspects, one or more potential splice donor and / or splice acceptor sites exhibit a splicing event score or splicing event probability 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 with a maximum of 1.0), and the site can be a candidate for splice site elimination or removal. In some aspects, the scores at one or more potential splice donor and / or splice sites, such as the scores used by GeneSplicer, are based on the difference between the log odds score of the sequence returned by a true Markov model and the score calculated by a false Markov model. In certain embodiments, splice donor sites and splice acceptor sites are evaluated independently or individually. In some embodiments, splice donor sites and splice acceptor sites are evaluated as a splice donor / acceptor pair.

[0335] In some embodiments, one or more splice donor and / or splice acceptor sites, such as potential splice donor and / or acceptor sites that may be involved in cryptic splicing events that are undesired or result in undesired RNA heterogeneity, are eliminated. In some embodiments, eliminating one or more splice sites comprises modifying (e.g., by substitution or replacement) one or more nucleotides in, at, containing, or near the splice donor and / or acceptor sites that are candidates for removal. In some aspects, specific nucleotides within the codon at, containing, or near the splice site are modified (e.g., by substitution or replacement). In some aspects, the modification (such as substitution or replacement) preserves or maintains the amino acid encoded by the specific codon at the site while removing the potential splice donor and / or acceptor site.

[0336] In some embodiments, the codons to be modified at or near the splice site comprise one or more codons that involve one or both of two nucleotides at a potential splice site (in some cases, referred to as "splice site codons"). When potential splicing is predicted to occur between two nucleotides in a codon, that codon is the sole splice site codon for that splice site. If potential splicing is predicted to occur between two adjacent codons, for example, between the last nucleotide of the first codon and the first nucleotide of the next codon, both of those codons are splice site codons. For example, for a splice site predicted to be at the boundary of two codons, both of the adjacent codons can be candidates for nucleotide modification. In some embodiments, the one or more codons comprise one splice site codon. In some embodiments, the one or more codons comprise two splice site codons. In some embodiments, potential splice donor sites are eliminated by modifying one or two splice site codons. In some embodiments, potential splice acceptor donor sites are eliminated by modifying one or two splice site codons. In some embodiments, one or both of the codons at the splice site are not modified, such as when there are no synonymous codons for the splice site codon. In some embodiments, if there are no synonymous codons available for a particular splice site codon, one or more nucleotides in an adjacent codon can be modified. In some embodiments, the one or more modified codons include a splice site codon, wherein the modification comprises changing one or both of the nucleotides at the splice site to one or more different nucleotides. In some embodiments, splice donor sites are eliminated by modifying one or two splice site codons, wherein the modification does not change one or both of the nucleotides at the splice site to different nucleotides, but adjacent nucleotides, such as a portion of a codon adjacent to the splice site, are modified. In some embodiments, the adjacent or neighboring nucleotides that can be modified include nucleotides that are part of an adjacent or neighboring codon that is one, two, three, four, five, six, seven, eight, nine, or ten codons upstream or downstream of the splice site codon.

[0337] In some cases, polynucleotides can be manually modified while preserving the encoded amino acid sequence to reduce the probability of predicted splice sites. In some embodiments, one or more of the predicted splice sites having a splice site probability of at least 80%, 85%, 90%, or 95% are manually modified to reduce the probability of splicing events. In some embodiments, one or more modifications are by nucleotide substitution or replacement of 1, 2, 3, 4, 5, 6, or 7 nucleotides. In some embodiments, the modification is at the junction of the splice donor site or at the junction of the splice acceptor site. In some embodiments, at least one of the one or more nucleotide modifications is 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 with reduced cryptic splice site probability can be generated. In some embodiments, the splice donor site and the splice acceptor site are evaluated as a splice donor / acceptor pair. In certain embodiments, the splice donor site and the splice acceptor site are evaluated independently or individually rather than as part of a splice donor / acceptor pair. In some embodiments, one or more of the predicted splice sites are not eliminated. In some embodiments, splice sites (such as known or predicted splice sites) within the promoter region of the transcript are not eliminated.

[0338] In some embodiments, one or more potential donor splice sites are eliminated by modifying one or two splice site codons or one or more neighboring or adjacent codons (e.g., if a synonymous codon for the splice site codon is not available). In some embodiments, one or more potential acceptor splice sites are eliminated by modifying one or two splice site codons or one or more neighboring or adjacent codons (e.g., if a synonymous codon for the splice site codon is not available). In some embodiments, neighboring or adjacent codons that are modified include codons within one, two, three, four, five, six, seven, eight, nine, or ten codons upstream or downstream of the splice site codon, such as codons within one, two, or three codons of the splice site. In some embodiments, a potential branch site for splicing is removed or eliminated. In some aspects, the nucleotides within the codons at or near the branch site may be modified, such as by substitution or replacement, to eliminate cryptic splicing and / or reduce RNA heterogeneity. In some embodiments, modifying one or more nucleotides may involve substituting or replacing one of the nucleotides that may be involved in splicing (such as at a splice donor site, a splice acceptor site, or a splice branch site) such that the amino acid encoded by the codon is maintained and the nucleotide substitution or replacement does not alter the polypeptide sequence encoded by the polynucleotide. In some cases, the third position in 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.1 above). In some embodiments, the modification includes replacing the codon with a synonymous codon used in the species (such as a human) into which the polynucleotide is introduced. In some embodiments, the species is a human. In some embodiments, one or more codons are replaced with the corresponding synonymous codon most frequently used in the species or a synonymous codon with a similar usage frequency (e.g., closest usage frequency) to the corresponding codon (see, e.g., Section II.1 above).

[0339] In some embodiments, after an initially proposed modification, the transgenic candidacy for splice site removal is evaluated. In some aspects, after modification and / or codon optimization, the proposed modification can be re-evaluated to assess the proposed modification 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 additional evaluations are performed on the sequence, such as the modified or corrected nucleic acid sequence, using the same or one or more other or additional splice site prediction tools to further evaluate splice site removal, such as cryptic splice site removal. In some aspects, the proposed modification for subsequent steps is considered and iterative optimization can be used. In some aspects, any one of the identification and / or modification steps can be repeated, for example until the heterogeneity of the transcript is reduced compared to the heterogeneity of the initially measured transcript. In some embodiments, another or different modification can be made after iterative evaluation and assessment, such as substituting a different nucleotide at the same codon or making a modification at a different position or codon. In some embodiments, correspondingly different synonymous codons can be used, such as using the second most commonly used codon in a particular species or a codon with a similar usage frequency (e.g., the next closest usage frequency) to the corresponding codon (see, for example, Section II.1 above).

[0340] In some aspects, the proposed modification can be further evaluated to, for example, assess whether the modification creates an unwanted or additional restriction site in the polynucleotide. In some aspects, an additional restriction site may not be desired and another or different modification (e.g., substituting a different nucleotide at the same codon or making a modification at a different position or codon) can be considered. In some aspects, specific restriction sites, such as designated restriction sites, are avoided. In some aspects, if the modification does not substantially reduce the splice site prediction score, an additional or alternative modification can be proposed. In some embodiments, after one or more iterations of these methods, 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%.

[0341] In some embodiments, a computer system can be used to perform one or more steps, tools, functions, processes, or scripts. In some embodiments, splice site prediction, evaluation, and modification for splice site elimination or removal can be performed by computer-implemented methods and / or by methods that include computer-implemented steps. In some embodiments, comparing a sequence to a known database, calculating a splice site prediction score, determining potential nucleotide modifications, codon optimization, and / or any iterative step can be implemented by a computer or using computer-implemented steps, tools, functions, processes, or scripts. In certain embodiments, a computer system is provided that includes a processor and memory, where the memory contains instructions operable to cause the processor to perform any one or more of the steps of the methods provided herein. In some embodiments, steps, functions, processes, or scripts are performed computationally, such as using one or more computer programs and / or via 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 steps: selecting a sequence, writing the sequence in FASTA format, loading a codon table (e.g., from www.kazusa.or.jp / codon), running GeneSplicer, loading the predictions, parsing the codons, determining overlaps in the predictions, identifying the second most highly used synonymous codons, checking for restriction sites, generating annotations, or evaluating additional codons. Certain steps can evaluate both the forward and reverse strands. In some aspects, previously annotated splice site modifications can also be considered for iterative optimization. In some embodiments, any one or more of the steps, functions, processes, or scripts can be repeated.

[0343] In some embodiments, the polynucleotides encoding the CARs provided herein or the constructs provided herein include modifications that remove one or more splice donor and / or acceptor sites, such modifications potentially resulting in splicing events and / or reduced expression and / or increased RNA heterogeneity. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:119. In some embodiments, the CAR is encoded by the nucleotide sequence shown in SEQ ID NO:120. 3. Other Features

[0344] Also provided are vectors containing polynucleotides and host cells containing these vectors, for example for making chimeric antigen receptors. Also provided are methods for making chimeric antigen receptors. The nucleic acid can encode a chimeric antigen receptor comprising an antibody VL region and / or VH region (such as an antibody light chain and / or heavy chain). The nucleic acid can encode one or more binding domains (such as a BCMA binding domain and a GPRC5D binding domain), and each of these chimeric antigen receptors comprises an antibody VL region and / or VH region (such as a BCMA binding domain and a GPRC5D binding domain). In another embodiment, one or more vectors (such as expression vectors) containing such polynucleotides are provided. In another embodiment, host cells containing such polynucleotides are provided. In one such embodiment, the host cell comprises a vector containing a nucleic acid encoding a chimeric antigen receptor (such as being transformed by the vector), and the chimeric antigen receptor comprises an antibody VH region. In another such embodiment, the host cell contains (such as being transformed by the following): (1) a vector containing a nucleic acid encoding a chimeric antigen receptor that comprises an antibody VL region and an antibody VH region; or (2) a vector that contains a nucleic acid encoding a chimeric antigen receptor containing a first antibody and a second antibody. In some embodiments, the host cell contains one or more vectors (such as being transformed by the one or more vectors), and the one or more vectors contain 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, a composition containing one or more such host cells is 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 these host cells can express more than one chimeric antigen receptor

[0345] Also provided is a method for preparing a bispecific chimeric antigen receptor that binds BCMA and GPRC5D. For recombinant production of chimeric receptors, nucleic acid sequences encoding chimeric receptor antibodies as described herein, for example, can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acid sequences can be easily isolated and sequenced using conventional procedures (such as by using oligonucleotide probes capable of specifically binding to genes encoding antibody heavy and light chains). In some embodiments, a method for preparing a bispecific chimeric antigen receptor is provided, wherein the method comprises culturing a host cell containing a nucleic acid sequence encoding an antibody (such as a BCMA binding domain and a GPRC5D binding domain) as provided above under conditions suitable for expressing the receptor.

[0346] In some embodiments, a method for preparing a cell composition containing cells expressing a bispecific chimeric antigen receptor is provided.

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

[0348] Exemplary eukaryotic cells that can be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44.Lec13 CHO cells and FUT8 CHO cells; PER. cells; and NSO cells. In some embodiments, the heavy and / or light chains of an antibody (e.g., the VH region and / or the VL region) can be expressed in yeast (see, e.g., U.S. Publication No. 2006 / 0270045A1). In some embodiments, a particular eukaryotic host cell is selected based on its ability to perform the desired post-translational modifications on the heavy and / or light chains (e.g., the VH region and / or the VL region). For example, in some embodiments, CHO cells produce polypeptides with a higher level of sialylation than the same polypeptides produced in 293 cells. In certain instances, immune cells such as human immune cells are used to express the provided polypeptides encoding chimeric antigen receptors. In some instances, the immune cells are T cells, such as CD4+ and / or CD8+ immune cells. III. Engineered Cells

[0349] Also provided are cells containing a recombinant receptor (e.g., a chimeric antigen receptor), such as engineered cells, wherein the recombinant receptor is a recombinant receptor comprising an extracellular domain containing both a GPRC5D binding domain and a BCMA binding domain as provided herein. Also provided are populations of such cells, compositions containing such cells and / or enriched for such cells, such as compositions in which the cells expressing the GPRC5D binding domain and the BCMA binding domain account for at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or a higher percentage of the total cells or a particular type of cells such as T cells, CD8+ cells or CD4+ cells in the composition.

[0350] Engineered cells (such as engineered cells) containing a recombinant receptor are also provided, and the recombinant receptor comprises 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 the GPRC5D binding domain included in the anti-GPRC5D CAR described in this application or the GPRC5D binding domain included in any other source (see, for example, WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245). Exemplary GPRC5D binding domains are described in Part I. The BCMA binding domain can be any known BCMA binding domain, such as the GPRC5D binding domain included in the anti-BCMACAR described in this application or the BCMA binding domain included in any other source (see, for example, WO 2013 / 154760, WO 2015 / 052538, WO 2015 / 090229, WO 2015 / 092024, WO 2015 / 158671, WO 2016 / 014565, WO 2016 / 014789, WO 2016 / 094304, WO 2016 / 166630, WO 2017 / 021450, WO 2017 / 083511, WO 2017 / 130223, WO 2017 / 211900, WO 2018 / 085690, WO 2018 / 028647). Exemplary BCMA binding domains are described in Part I.

[0351] In some embodiments, the engineered cells provided herein can be combined with one or more populations of engineered cells expressing one or more other recombinant receptors. Such populations of engineered cells can be formulated as the same or separate compositions. Among these compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering to a subject, such as a patient, any of the cells and compositions provided herein.

[0352] Accordingly, genetically engineered cells expressing recombinant receptors containing antibodies are also provided, such as cells containing CARs. The cells are generally eukaryotic cells, such as mammalian cells, and are 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 innate or adaptive immune cells, for example, bone marrow or lymphocytes, including 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). In some embodiments, the cells include T cells. The cells are typically primary cells, such as cells isolated directly from a subject and / or cells isolated from a subject and cryopreserved. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as a complete T cell population, CD4+ cells, CD8+ cells, and their subsets, such as cells defined by the following: function, activation state, maturity, differentiation potential, expansion, recirculation, location, and / or persistence, 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+ T cells and CD8+ T cells. When referring to a subject to be treated, the cells can be allogeneic cells and / or autologous cells. Among these methods are off-the-shelf methods. In some aspects, such as in off-the-shelf technology, the cells are multipotent and / or pluripotent cells, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, these methods include isolating cells from a subject as described herein, preparing, processing, culturing, and / or genetically engineering them, and reintroducing them into the same patient before or after cryopreservation.

[0353] Subtypes and subsets of T cells and / or CD4+ T cells and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, 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, naturally occurring 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; α / β T cells and δ / γ T cells.

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

[0355] In some embodiments, the cells comprise one or more polynucleotides introduced by genetic engineering and thereby express a recombinant product or a genetically engineered product of such polynucleotides. In some embodiments, the polynucleotide is a heterologous polynucleotide, i.e., a polynucleotide that is not normally present in the cell or in a sample obtained from the cell, such as a polynucleotide obtained from another organism or cell, and such polynucleotides are not typically found, for example, in engineered cells and / or in the organism from which such cells are derived. In some embodiments, the polynucleotide is not a naturally occurring polynucleotide, such as a polynucleotide not found in nature, including polynucleotides that contain chimeric combinations of polynucleotides 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.) as described herein, such as a vector containing a nucleic acid encoding a recombinant receptor as described herein. A. Vectors and Methods for Genetic Engineering

[0356] Also provided are methods, polynucleotides, compositions, and kits for expressing a bispecific recombinant receptor (e.g., a CAR) and for manufacturing genetically engineered cells that express such receptors. In some embodiments, one or more recombinant receptors (e.g., a CAR) can be genetically engineered into a cell or cells. Genetic engineering generally involves introducing a nucleic acid encoding a recombinant or engineered component into a cell, 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 the cells with a stimulus that induces a response, such as proliferation, survival, and / or activation, as measured, for example, by the expression of a cytokine or an activation marker; subsequently transducing the activated cells, and expanding in culture to a number sufficient for clinical applications.

[0358] In some cases, overexpression of stimulatory factors (such as lymphokines or cytokines) can be toxic to a subject. Thus, in some cases, engineered cells include genetic segments that render the cells liable to undergo negative selection in vivo when administered, such as in adoptive immunotherapy. For example, in some aspects, the cells are genetically engineered such that they can be eliminated due to changes in the in vivo conditions of the patient to whom they are administered. A phenotype that can be negatively selected can be generated due to the insertion of genes that confer sensitivity to an administered agent (such as a compound). Genes that can be negatively selected include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers sensitivity to ganciclovir (Wigler et al., Cell 2:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, the bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0359] In some aspects, the cells are further genetically engineered to promote the expression of cytokines or other factors. Various methods for introducing genetically engineered constructs, such as antigen receptors, such as CARs, are well known and can be used in conjunction with the provided methods and compositions. Exemplary methods include methods for transferring polynucleotides encoding these receptors, including via viral (such as retroviral or lentiviral) transduction, transposons, and electroporation.

[0360] In some embodiments, recombinant infectious viral particles, such as vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV), are used to transfer recombinant polynucleotides into cells. In some embodiments, recombinant lentiviral vectors, such as HIV-1 lentivirus-based vectors (lentiviral vectors; see, for example, Amado et al., Science. July 30, 1999; 285(5428):674-676), or retroviral vectors such as gamma-retroviral vectors (see, for example, Koste et al. (2014) Gene Therapy April 3, 2014. 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. November 29, 2011(11):550-557) are used to transfer recombinant polynucleotides into T cells.

[0361] In some embodiments, the retroviral vector 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 self-inactivating (SIN). In some embodiments, the vector is a conditionally replicating (movable) 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 includes those derived from any avian or mammalian cell source. Lentiviruses or retroviruses are generally amphotropic, meaning they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol, and / or env sequences. Lentiviral transduction methods 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 variet...

Claims

1. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino-terminus to the 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) 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.

2. The bispecific CAR according to claim 1, wherein the extracellular domain sequentially comprises, from the amino-terminus to the carboxy-terminus, 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.

3. The bispecific CAR according to claim 1 or claim 2, wherein the extracellular domain sequentially comprises, from the amino-terminus to the 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.

4. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino-terminus to the 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.

5. The bispecific CAR according to claim 1 or claim 2, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

6. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

7. The bispecific CAR according to claim 1 or claim 2, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

8. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

9. The bispecific CAR according to claim 1 or claim 2, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

10. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino-terminus to the 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.

11. The bispecific CAR according to claim 1, wherein the extracellular domain sequentially comprises, 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 claim 11, wherein the extracellular domain sequentially comprises, from the amino-terminus to the 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.

13. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, wherein the GPRC5D-binding domain comprises a heavy chain variable (VH) region and a light chain variable (VL) region; the BCMA-binding domain comprises a VH region and a VL region, and wherein the extracellular domain sequentially comprises, from the amino-terminus to the 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.

14. The bispecific CAR according to claim 1 or claim 11, wherein the extracellular domain sequentially comprises, from the amino-terminus to the 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.

15. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

16. The bispecific CAR according to claim 1 or claim 11, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

17. A bispecific chimeric antigen receptor comprising: (a) An extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

18. The bispecific CAR according to claim 1 or claim 11, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

19. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy-chain variable (VH) region and a light-chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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.

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

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

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

23. The bispecific CAR according to any one of claims 20-22, 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. The bispecific CAR according to any one of claims 1-23, wherein: (a) the VH region and the VL region of the GPRC5D-binding domain are connected by a linker; or (b) the VH region and the VL region of the BCMA-binding domain are connected by a linker.

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 bispecific chimeric antigen receptor (CAR) comprising: (a) an extracellular domain, the extracellular domain comprising a GPRC5D-binding domain that binds to GPRC5D and a BCMA-binding domain that binds to BCMA, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises from the amino terminus to the carboxyl terminus: (i) the VH region of the GPRC5D-binding domain; (ii) the linker shown in SEQ ID NO:21; (iii) the VL region of the BCMA-binding domain; (iv) the linker shown in SEQ ID NO:17; (v) the VH region of the BCMA-binding domain; (vi) the linker shown 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.

27. A bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain, said extracellular domain comprising (i) a GPRC5D-binding domain that binds to GPRC5D, said GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) a BCMA-binding domain that binds to BCMA, said BCMA-binding domain comprising a VH region and a VL region, wherein said extracellular domain sequentially comprises, from the amino terminus to the carboxyl 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) An intracellular signaling domain.

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

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

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

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

32. The bispecific CAR according to any one of claims 29-31, 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 27-32, wherein the VH region and the 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 bispecific chimeric antigen receptor (CAR) comprising: (a) An extracellular domain, the extracellular domain comprising (i) a GPRC5D-binding domain that binds to GPRC5D, the GPRC5D-binding domain comprising a heavy chain variable (VH) region and a light chain variable (VL) region; and (ii) a BCMA-binding domain that binds to BCMA, the BCMA-binding domain comprising a VH region and a VL region, wherein the extracellular domain sequentially comprises, from the amino terminus to the carboxyl terminus: the VH region of the GPRC5D-binding domain; the VL region of the GPRC5D-binding domain; one 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 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 shown in SEQ ID NO:19 or SEQ ID NO:

21.

35. The bispecific CAR according to any one of claims 1-34, wherein the VH region of the GPRC5D-binding domain comprises CDR-1, CDR-2 and CDR-3, and the CDR-1, CDR-2 and CDR-3 comprise 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 1-35, wherein the VL region of the GPRC5D-binding domain comprises CDR-1, CDR-2 and CDR-3, and the CDR-1, CDR-2 and CDR-3 comprise 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 1-36, wherein the VH region of the GPRC5D-binding domain comprises CDR-1, CDR-2 and CDR-3 containing the amino acid sequences shown 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 containing the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

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

7.

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

8.

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

8.

41. The bispecific CAR according to any one of claims 1-40, 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 1-41, 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 1-42, 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 1-43, wherein the VH region of the BCMA-binding domain comprises CDR-1, CDR-2, and CDR-3 containing the amino acid sequences shown 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 1-44, wherein the VL region of the BCMA-binding domain comprises CDR-1, CDR-2, and CDR-3 containing 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 1-45, wherein the VH region of the BCMA-binding domain comprises an amino acid sequence having at least about 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

15.

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

16.

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

16.

49. The bispecific CAR according to any one of claims 1-48, 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 1-49, 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 1-50, 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, 20-25, and 35-51, wherein the extracellular binding domain comprises the amino acid sequence shown in any one of SEQ ID NO: 77, 78, 79, and 80.

53. The bispecific CAR according to any one of claims 1, 20-25, and 35-51, wherein the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO: 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, 20-26, 35-51, and 53, wherein the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:

83.

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

84.

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

87.

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

81.

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

85.

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

86.

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

90.

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

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

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

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

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

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

25.

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

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

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

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

27.

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

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

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

28.

74. The bispecific CAR according to any one of claims 1-73, wherein the intracellular signaling domain is a domain 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 1-74, wherein the intracellular signaling domain comprises the cytoplasmic signaling domain of the CD3-ζ chain, optionally the human CD3-ζ chain.

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

30.

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

78. The bispecific CAR according to claim 77, wherein the co-stimulatory signaling region is located between the transmembrane region and the intracellular signaling domain.

79. The bispecific CAR according to claim 77 or 78, wherein the co-stimulatory signaling region comprises the intracellular signaling domain or a signaling portion thereof of a T cell co-stimulatory molecule.

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

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

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

29.

83. The bispecific CAR according to any one of claims 1-82, wherein 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.

84. The bispecific CAR according to any one of claims 1-83, wherein the CAR comprises 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 according to claim 84, wherein the CAR comprises the amino acid sequence shown in SEQ ID NO:

37.

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

87. The bispecific CAR according to claim 86, wherein the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:

83.

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

37.

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

119.

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

91. The bispecific CAR according to claim 90, wherein the extracellular binding domain comprises the amino acid sequence shown in SEQ ID NO:

86.

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

40.

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

120.

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

95. The polynucleotide according to claim 94, wherein the polynucleotide comprises the nucleotide sequences shown in SEQ ID NO:105-120.

96. A polynucleotide comprising any one of the nucleotide sequences shown in SEQ ID NO:105-120.

97. The polynucleotide according to any one of claims 94-96, wherein the polynucleotide is optimized by splice site elimination.

98. The polynucleotide according to any one of claims 94-97, wherein the polynucleotide is codon-optimized for expression in human cells.

99. The polynucleotide according to any one of claims 94-98, wherein the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:119 or SEQ ID NO:

120.

100. A polynucleotide according to any one of claims 94-99, said polynucleotide comprising the nucleotide sequence shown in SEQ ID NO:

119.

101. A polynucleotide according to any one of claims 94-100, said polynucleotide comprising the nucleotide sequence shown in SEQ ID NO:

120.

102. A vector, said vector comprising a polynucleotide according to any one of claims 94-101.

103. The vector according to claim 102, which is a viral vector.

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

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

106. A cell, said cell comprising a CAR according to any one of claims 1-93.

107. A cell, said cell comprising a polynucleotide according to any one of claims 90-101 or a vector according to any one of claims 102-105.

108. The cell according to claim 106 or claim 107, wherein said cell is an immune cell.

109. The cell according to any one of claims 106-108, wherein said cell is a lymphocyte.

110. The cell according to any one of claims 106-109, wherein said cell is a NK cell or a T cell.

111. The cell according to any one of claims 106-110, wherein said cell is a T cell.

112. The cell according to claim 111, wherein said T cell is a CD4+ T cell or a CD8+ T cell.

113. The cell according to claim 111 or claim 112, wherein said T cell is a primary T cell.

114. The cell according to claim 106 or claim 107, wherein said cell is a stem cell.

115. The cell according to claim 114, wherein said stem cell is a multipotent and pluripotent stem cell.

116. The method according to claim 114 or claim 115, wherein said stem cell is an induced pluripotent stem cell (iPSC).

117. The cell according to any one of claims 106-112, wherein said cell has differentiated from an induced pluripotent stem cell.

118. The cell according to any one of claims 106-117, wherein said cell is an allogeneic cell.

119. The cell according to any one of claims 106-118, wherein said cell is engineered to be immune-deficient.

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

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

122. The pharmaceutical composition according to claim 121, which further comprises a pharmaceutically acceptable excipient.

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

124. The composition according to any one of claims 121 - 123, wherein the composition comprises CD4+ T cells and CD8+ T cells.

125. The composition according to claim 124, wherein the composition comprises a ratio of CD4+ T cells to CD8+ T cells, the ratio being between about 1:3 and about 3:1, optionally between about 1:2 and about 2:1, and further optionally about 1:

1.

126. The composition according to claim 124 or 125, wherein the composition comprises a ratio of CD4+ T cells to CD8+ T cells, the ratio being between about 1:3 and about 3:

1.

127. The composition according to any one of claims 124 - 126, wherein the composition comprises a ratio of CD4+ T cells to CD8+ T cells, the ratio being about 1:

1.

128. The composition according to 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. The composition according to 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 the cells in the composition express the CAR.

130. The composition according to any one of claims 121 - 129, wherein, among the plurality of cells expressing the CAR in the composition, 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 tonic signaling.

131. The composition according to any one of claims 121 - 130, wherein the composition comprises between about 1.0x10 7 CAR-expressing T cells and 1.2x10 9 CAR-expressing T cells, between about 1.0x10 7 CAR-expressing T cells and 6.5x10 8 CAR-expressing T cells, between about 1.5x10 7 CAR-expressing T cells and 6.5x10 8 CAR-expressing T cells, between about 1.5x10 7 CAR-expressing T cells and 6.0x10 8 CAR-expressing T cells, between about 2.5x10 7 CAR-expressing T cells and 6.0x10 8 CAR-expressing T cells, between about 5.0x10 7 CAR-expressing T cells and 6.0x10 8 CAR-expressing T cells, between about 1.25x10 7 CAR-expressing T cells and 1.2x10 9 CAR-expressing T cells, between about 1.5x10 7 CAR-expressing T cells and 1.2x10 9 CAR-expressing T cells, between about 5.0x10 7 CAR-expressing T cells and 4.5x10 8 CAR-expressing T cells, or between about 1.5x10 8 CAR-expressing T cells and 3.0x10 8 CAR-expressing T cells, with both end values included.

132. The composition according to any one of claims 121-131, wherein the composition comprises about 1.5x10 7 cells, about 2.5x10 7 cells, about 5.0x10 7 cells, about 7.5x10 7 cells, about 1.0x10 8 cells, about 1.25x10 8 cells, about 1.5x10 8 cells, about 1.75x10 8 cells, about 2x10 8 cells, about 2.25x10 8 cells, about 2.5x10 8 cells, about 3.0x10 8 cells, about 3.5x10 8 cells, about 4x10 8 cells, about 4.5x10 8 cells, about 6.0x10 8 cells, about 8.0x10 8 , or about 1.2x10 9 CAR-expressing T cells.

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

134. The method according to claim 133, wherein the cells are administered to the subject at a dose of from or about 1x10 7 CAR-expressing T cells to 1x10 9 CAR-expressing T cells.

135. The method according to claim 133, wherein the cells are administered to the subject at a dose of from or about 2.5x10 7 CAR-expressing T cells to about 4.5x10 8 CAR-expressing T cells.

136. The method according to any one of claims 133-135, wherein the cells are administered to the subject at a dose of or about 2.5x10 7 CAR-expressing T cells.

137. The method according to any one of claims 133-135, wherein the cells are administered to the subject at a dose of about 7.5x10 7 CAR-expressing T cells.

138. The method according to any one of claims 133-135, wherein the cells are administered to the subject at a dose of 8 CAR-expressing T cells that is about 1.5x10 8 per.

139. The method according to any one of claims 133 - 135, wherein the cells are administered to the subject at a dose of about 3.0x10 8 CAR-expressing T cells.

140. The method according to any one of claims 133-135, wherein the cells are administered to the subject at a dose of 8 CAR-expressing T cells of or about 4.5x10 8 per subject.

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

142. The method according to any one of claims 133 - 141, wherein the lymphodepletion therapy is completed within about 7 days before the start of the administration of the dose of the CAR-expressing T cells.

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

144. The method according to any one of claims 133 - 143, wherein the lymphodepletion therapy comprises the administration of fludarabine and / or cyclophosphamide.

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

146. The method according to any one of claims 133 - 145, wherein the lymphodepletion therapy comprises administering cyclophosphamide at or about 200 - 400 mg / m 2 per day, inclusive of the end values.

147. The method according to any one of claims 133 - 146, wherein the lymphodepletion therapy comprises administering cyclophosphamide at or about 300 mg / m 2 per day.

148. The method according to any one of claims 133 - 145, wherein the lymphodepletion therapy comprises administering fludarabine at or about 20 - 40 mg / m 2 per day, the end values being included.

149. The method according to any one of claims 133 - 146 and 148, wherein the lymphodepletion therapy comprises administering fludarabine at or about 30 mg / m 2 per day.

150. The method according to any one of claims 133 - 149, wherein the lymphodepletion therapy comprises the administration of fludarabine and cyclophosphamide for 2 - 4 days.

151. The method according to any one of claims 133 - 150, wherein the lymphodepletion therapy comprises the administration of fludarabine and cyclophosphamide for 3 days.

152. The method according to any one of claims 133 - 143, wherein the lymphodepletion therapy comprises the administration of bendamustine.

153. The method according to any one of claims 133 - 143 and 152, wherein the lymphodepletion therapy comprises administering bendamustine at or about 50 - 130 mg / m 2 per day, the end values being included.

154. The method according to any one of claims 133 - 143, 152 and 153, wherein the lymphodepletion therapy comprises administering bendamustine at or about 90 mg / m 2 per day.

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

156. The method according to any one of claims 133 - 143 and 152 - 155, wherein the lymphodepletion therapy comprises the administration of bendamustine for 2 days.

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

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

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

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

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

162. The method according to any one of claims 133 - 161, wherein, the subject has received at least 1 but no more than 3 prior therapies.

163. The method according to claim 161 or claim 162, wherein the prior therapy is a proteasome inhibitor, an immunomodulatory agent, an anti - CD38 antibody, a prior therapy comprising autologous hematopoietic stem cell transplantation (HSCT), or any combination thereof.

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

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

166. The use according to claim 164 or claim 165, wherein the disease or disorder is cancer, optionally a plasma cell malignancy.

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

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

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

170. The use according to any one of claims 164 - 169, wherein the subject has received one or more prior therapies.

171. The use according to any one of claims 164 - 169, wherein, the subject has received at least 1 but no more than 3 prior therapies.

172. The use according to claim 170 or claim 171, wherein the prior therapy is a proteasome inhibitor, an immunomodulatory agent, an anti - CD38 antibody, a prior therapy comprising autologous hematopoietic stem cell transplantation (HSCT), or any combination of the foregoing.

173. The cell according to any one of claims 106 - 120 or the composition according to any one of claims 121 - 132, for treating a disease or disorder of a subject.

174. The cell or composition according to claim 173, wherein the disease or disorder is cancer, optionally a plasma cell malignancy.

175. The cell or composition according to claim 173 or claim 174, wherein the disease or disorder is a cancer expressing BCMA and / or a cancer expressing GPRC5D.

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

177. The cell or composition according to any one of claims 173 - 176, wherein the disease or disorder is relapsed / refractory multiple myeloma (RRMM).

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

179. The use according to any one of claims 173 - 178, wherein, the subject has received at least 1 but no more than 3 prior therapies.

180. The cell or composition according to claim 178 or 179, wherein the prior therapy is a proteasome inhibitor, an immunomodulatory agent, an anti - CD38 antibody, a prior therapy comprising autologous hematopoietic stem cell transplantation (HSCT), or any combination of the foregoing.

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

182. The kit according to claim 181, wherein the manual specifically states to administer the CAR, the cell or the composition to a subject suffering from a disease or disorder.

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

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