Combination therapies with cell therapies expressing a CAR targeting GPRC5D and related methods and uses

Abstract

Provided herein are methods and uses of combination therapies involving a GPRC5D-targeted cell therapy comprising a chimeric antigen receptor (CAR) containing an extracellular antigen binding domain that binds to G Protein-Coupled Receptor Class C Group 5 Member D (GPRC5D) and a combination agent, e.g., a bispecific antibody, for treating a subject having a cancer, such as multiple myeloma, as well as related methods, uses, and articles of manufacture.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 538,264, filed September 13, 2023, entitled “COMBINATION THERAPIES WITH A CELL THERAPY EXPRESSING A GPRC5D-TARGETING CAR AND RELATED METHODS AND USES,” the contents of which are incorporated herein by reference in their entirety. By referencing and incorporating into the sequence list

[0002] This application is submitted together with an electronic sequence list. The sequence list is provided as a file with the title 735042027640SeqList.XML, created on August 28, 2024, and has a size of 345,518 bytes. Information from the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates in several aspects to methods and uses of combination therapies involving T-cell therapies (e.g., CAR T-cell therapy) and additional agents (e.g., bispecific antibodies; e.g., T-cell connectors targeting BCMA) for treating subjects with cancers such as multiple myeloma, as well as related methods, uses, and articles thereof. T-cell therapies include cells expressing recombinant receptors (e.g., chimeric antigen receptors (CARs) containing an extracellular antigen-binding domain that binds to a G protein-coupled receptor class C5 member D (GPRC5D)). Background Technology

[0004] G protein-coupled receptor class C5 member D (GPRC5D) is a G protein-coupled receptor whose specific function is not yet determined. GPRC5D expression is significantly higher in bone marrow samples from patients with multiple myeloma (MM) compared to the lowest expression found in bone marrow samples from patients with other hematologic malignancies. Based on its expression, GPRC5D could be a marker and therapeutic target for MM tumors. GPRC5D-binding chimeric antigen receptors (CARs) and cells expressing such CARs are available. However, there is still a need for improved GPRC5D-binding CARs, engineered targeted cells expressing GPRC5D-CARs, and combination therapies. This article provides implementation schemes to meet these needs. Summary of the Invention

[0005] This article provides a method for treating relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering CAR-T cell therapy to a subject with RRMM, the engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D); and (2) administering to the subject a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3.

[0006] In some embodiments, the bispecific antibody is administered using a dosing regimen that begins no earlier than 60 days after administration of the CAR T-cell therapy. In some embodiments, the bispecific antibody comprises at least one Fab fragment of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody. In some embodiments, the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody.

[0007] In some embodiments, at least one fixed dose of the bispecific antibody is administered in an amount between 1 mg and 50 mg. In some embodiments, the dosing regimen of the bispecific antibody is a cyclical regimen comprising more than one treatment cycle. In some embodiments, each treatment cycle is a 28-day cycle. In some embodiments, the dosing regimen of the bispecific antibody includes a first treatment cycle (C1), which is an escalating dosing regimen comprising one or more start doses and one or more maintenance doses, wherein the maintenance dose is greater than the start dose. In some embodiments, C1 further includes one or more intermediate doses, wherein the intermediate dose is greater than the start dose and less than the maintenance dose.

[0008] This article provides a method for treating relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering CAR-T cell therapy to a subject with RRMM, the engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D); and (2) administering to the subject a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3 in a dosing regimen initiated after the CAR-T cell administration; wherein the dosing regimen of the bispecific antibody comprises a cyclical regimen comprising more than one treatment cycle, wherein each treatment cycle is a 28-day treatment cycle, and wherein the first treatment cycle (C1) is an escalating dosing regimen comprising: (i) an initiation phase, wherein the subject is administered one or more initiation doses of the bispecific antibody; (ii) an intermediate phase, wherein the subject is administered one or more intermediate doses of the bispecific antibody, wherein the intermediate dose is greater than the initiation dose; and (iii) The maintenance phase involves administering one or more maintenance doses of the bispecific antibody to the subject, wherein each maintenance dose is greater than the intermediate dose.

[0009] In some embodiments, the bispecific antibody comprises at least one Fab fragment of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody. In some embodiments, the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody. In some embodiments, administration of the bispecific antibody begins 28 days to 6 months after administration of the CAR-T cell therapy to the subject. In some embodiments, administration of the bispecific antibody begins no earlier than 60 days after the CAR-T cell therapy. In some embodiments, administration of the bispecific antibody begins at or approximately 60 days after the CAR-T cell therapy.

[0010] In some embodiments, the initial phase includes a single starting dose. In some embodiments, the starting dose is about 1.0 mg to 6 mg. In some embodiments, the starting dose is about 3 mg. In some embodiments, the intermediate phase includes a single intermediate dose. In some embodiments, the intermediate dose is about 4.5 mg to 10 mg. In some embodiments, the intermediate dose is about 6.0 mg. In some embodiments, the maintenance dose is about 6 mg to about 50 mg. In some embodiments, the maintenance dose is about 25.0 mg to 35.0 mg. In some embodiments, the maintenance dose is about 30 mg. In some embodiments, the maintenance dose is about 6 mg to 18 mg. In some embodiments, the maintenance dose is about 10 mg.

[0011] In some embodiments, the one or more starting doses are administered during the first week of C1. In some embodiments, a single starting dose is administered on day 1 (D1) of C1. In some embodiments, the one or more intermediate doses are administered during the first week of C1. In some embodiments, a single intermediate dose is administered on one of days 3-6 of C1. In some embodiments, the single intermediate dose is administered on day 4 (D4) of C1. In some embodiments, the one or more maintenance doses are administered weekly after the first week of C1. In some embodiments, the one or more maintenance doses are administered on days 8 (D8), 15 (D15), and 22 (D22) of C1. In some embodiments, the bispecific antibody is administered on day 1 at about 3 mg, on day 4 at about 6 mg, and on days 8, 15, and 22 at about 30 mg in C1.

[0012] In some embodiments, in C1, the bispecific antibody is administered at approximately 3 mg on day 1, approximately 6 mg on day 4, and approximately 10 mg on days 8, 15, and 22. In some embodiments, the dosing regimen further comprises two or more consecutive treatment cycles, each cycle comprising a once-weekly dosing of the maintenance dose (Q1W). In some embodiments, the dosing regimen comprises 2-4 once-weekly treatment cycles. In some embodiments, the dosing regimen comprises a second treatment cycle (C2) comprising a once-weekly dosing of the maintenance dose. In some embodiments, the dosing regimen comprises a second treatment cycle (C2) and a third treatment cycle (C3), wherein each of C2 and C3 is characterized by a once-weekly dosing of the maintenance dose. In some embodiments, the once-weekly dosing of the maintenance dose is administered on days 1, 8, 15, and 22. In some embodiments, C2 comprises administering the bispecific antibody at approximately 30 mg on days 1, 8, 15, and 22, and C3 comprises administering the bispecific antibody at approximately 30 mg on days 1, 8, 15, and 22. In some embodiments, C2 includes administering the bispecific antibody at about 10 mg on days 1, 8, 15, and 22, and C3 includes administering the bispecific antibody at about 10 mg on days 1, 8, 15, and 22.

[0013] In some embodiments, after a monthly treatment cycle, the dosing regimen includes one or more additional treatment cycles characterized by a reduced frequency of administration of the maintenance dose of the bispecific antibody. In some embodiments, the one or more additional treatment cycles include dosing every other week (Q2W), once a month (Q4W), or a combination thereof. In some embodiments, the one or more additional treatment cycles include one or more treatment cycles of administering the maintenance dose for Q2W. In some embodiments, the dosing regimen includes 2-6 treatment cycles of administering the maintenance dose for Q2W. In some embodiments, the dosing regimen includes 2-4 treatment cycles of administering the maintenance dose for Q2W. In some embodiments, the dosing regimen includes 3 treatment cycles of administering the maintenance dose for Q2W. In some embodiments, the dosing regimen includes a fourth treatment cycle (C4), a fifth treatment cycle (C5), and a sixth treatment cycle (C6), wherein each of C4, C5, and C6 is characterized by administration of the maintenance dose every other week (Q2W).

[0014] In some embodiments, the maintenance dose is administered Q2W on days 1 and 15. In some embodiments, C4 comprises administering the bispecific antibody at approximately 30 mg on days 1 and 15, C5 comprises administering the bispecific antibody at approximately 30 mg on days 1 and 15, and C6 comprises administering the bispecific antibody at approximately 30 mg on days 1 and 15. In some embodiments, C4 comprises administering the bispecific antibody at approximately 10 mg on days 1 and 15, C5 comprises administering the bispecific antibody at approximately 10 mg on days 1 and 15, and C6 comprises administering the bispecific antibody at approximately 10 mg on days 1 and 15. In some embodiments, the one or more additional treatment cycles further comprise administering the maintenance dose at one or more Q4Ws following one or more Q2Ws of administration. In some embodiments, the dosing regimen comprises 4-10 treatment cycles of administering the maintenance dose at Q4Ws.

[0015] In some embodiments, the dosing regimen comprises 5-8 treatment cycles of administering the maintenance dose Q4W. In some embodiments, the dosing regimen comprises 6 treatment cycles of administering the maintenance dose Q4W. In some embodiments, the dosing regimen comprises a seventh treatment cycle (C7), an eighth treatment cycle (C8), a ninth treatment cycle (C9), a tenth treatment cycle (C10), an eleventh treatment cycle (C11), and a twelfth treatment cycle (C12), wherein each of C7, C8, C9, C10, C11, and C12 is characterized by once-monthly (Q4W) administration of the maintenance dose. In some embodiments, the maintenance dose Q4W is administered on day 1 of each cycle. In some embodiments, the bispecific antibody is administered to the subject for up to 12 treatment cycles.

[0016] This article also provides a method for treating relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering CAR-T cell therapy to a subject with RRMM, the engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D); (2) administering to the subject a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3, wherein the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody, and wherein the bispecific antibody is administered in a dosing regimen that begins no earlier than 60 days after the CAR T cell administration, and the dosing regimen is characterized by 12 treatment cycles, each lasting 28 days; wherein: (i) the first treatment cycle of the bispecific antibody administration comprises administering 3 mg on day 1, 6 mg on day 4, and 30 mg on days 8, 15, and 22; (ii) The second and third treatment cycles of the bispecific antibody each consist of administering 30 mg on days 1, 8, 15 and 22; (iii) the fourth, fifth and sixth treatment cycles of the bispecific antibody each consist of administering 30 mg on day 1 and day 15; (iv) the 7th to 12th treatment cycles of the bispecific antibody each consist of administering 30 mg on day 1.

[0017] This article also provides a method for treating relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) administering CAR-T cell therapy to a subject with RRMM, the engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D); (2) administering to the subject a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3, wherein the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody, and wherein the bispecific antibody is administered in a dosing regimen that begins no earlier than 60 days after the administration of the CAR T cells, and wherein the dosing regimen is characterized by 12 treatment cycles, each lasting 28 days, wherein: (i) the first treatment cycle of the bispecific antibody comprises administering 3 mg on day 1, 6 mg on day 4, and 10 mg on days 8, 15, and 22; (ii) The second and third treatment cycles of the bispecific antibody each consist of administering 10 mg on days 1, 8, 15, and 22; (iii) the fourth, fifth, and sixth treatment cycles of the bispecific antibody each consist of administering 10 mg on days 1 and 15; and (iv) the 7th to 12th treatment cycles of the bispecific antibody each consist of administering 10 mg on day 1.

[0018] In some embodiments, the bispecific antibody is administered subcutaneously. In some embodiments, the bispecific antibody is a trivalent bispecific antibody comprising two Fab fragments of an anti-BCMA antibody, one Fab fragment of an anti-CD3 antibody, and an Fc moiety. In some embodiments, the bispecific antibody is in the form of BCMA Fab-Fc-CD3 Fab-BCMA Fab. In some embodiments, the anti-BCMA Fab comprises: (a) a heavy chain variable region (VH) containing the VH sequence shown in SEQ ID NO: 210, comprising CDR1, CDR2, and CDR3, and a light chain variable region (VL) containing the VL sequence shown in SEQ ID NO: 216; (b) a VH containing the VH sequence shown in SEQ ID NO: 210, comprising CDR1, CDR2, and CDR3, and a VL containing the VL sequence shown in SEQ ID NO: 217; (c) a VH containing the VH sequence shown in SEQ ID NO: 210, comprising CDR1, CDR2, and CDR3, and a VL containing the VL sequence shown in SEQ ID NO: 218; (d) a VH containing the VH sequence shown in SEQ ID NO: 211, comprising CDR1, CDR2, and CDR3, and a VL containing the VH sequence shown in SEQ ID NO: 218. (e) VL containing CDR1, CDR2, and CDR3 of the VL sequence shown in SEQ ID NO: 216; (f) VH containing CDR1, CDR2, and CDR3 of the VH sequence shown in SEQ ID NO: 212 and VL containing CDR1, CDR2, and CDR3 of the VL sequence shown in SEQ ID NO: 216; or (g) VH containing CDR1, CDR2, and CDR3 of the VH sequence shown in SEQ ID NO: 214 and VL containing CDR1, CDR2, and CDR3 of the VL sequence shown in SEQ ID NO: 215.

[0019] In some embodiments, the anti-BCMA Fab comprises: (a) a VH containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 225, 226, and 221, respectively; and a VL containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 227, 228, and 224, respectively; (b) a VH containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 225, 226, and 221, respectively; and a VL containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 229, 230, and 224, respectively; (c) a VH containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 225, 226, and 221, respectively; and a VL containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 231, 232, and 224, respectively; and (d) VH containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 233, 234, and 221 respectively; and VL containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 235, 236, and 237 respectively; (e) VH containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 238, 239, and 221 respectively; and VL containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 235, 236, and 237 respectively; (f) VH containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 240, 241, and 221 respectively; and VL containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 235, 236, and 237 respectively; or (g) VH containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 238, 239, and 221 respectively; and VL containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 235, 236, and 237 respectively; VHs containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 219, 220, and 221; and VLs containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 222, 223, and 224, respectively.

[0020] In some embodiments, the anti-BCMA Fab comprises a VH containing CDR1, CDR2, and CDR3 of the VH sequence shown in SEQ ID NO: 210, and a VL containing CDR1, CDR2, and CDR3 of the VL sequence shown in SEQ ID NO: 218. In some embodiments, the anti-BCMA Fab comprises a VH containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 225, 226, and 221, respectively; and a VL containing CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 231, 232, and 224, respectively.

[0021] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment comprises a VH containing the VH sequence shown in SEQ ID NO: 242, comprising CDR1, CDR2, and CDR3, and a VL containing the VL sequence shown in SEQ ID NO: 243, comprising CDR1, CDR2, and CDR3, respectively. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment comprises a VH containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 244, 245, and 246, respectively; and a VL containing the CDR1, CDR2, and CDR3 sequences shown in SEQ ID NO: 247, 248, and 249, respectively.

[0022] In some embodiments, the bispecific antibody comprises a combination of the heavy and light chains of the sequences shown in SEQ ID NO: 250, SEQ ID NO: 251, and SEQ ID NO: 252, and two copies of the sequence shown in SEQ ID NO: 253.

[0023] In some implementations, the bispecific antibody is anuctamab.

[0024] In some embodiments, the CAR comprises (1) an extracellular antigen-binding domain that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D), (2) an immunoglobulin hinge spacer of at least 125 amino acids in length; (3) a transmembrane domain; and (4) an intracellular signal transduction region comprising a cytoplasmic signal transduction domain of the CD3-ζ (CD3ζ) chain and a co-stimulatory signal transduction region; wherein the extracellular antigen-binding domain comprises V H District and V L Zone, wherein: the V HThe region contains the amino acid sequence shown in SEQ ID NO: 1 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% identity with SEQ ID NO: 1; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 2 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% identity with SEQ ID NO: 2; the V H The region contains the amino acid sequence shown in SEQ ID NO: 1 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% identity with SEQ ID NO: 1; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 116 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% identity with SEQ ID NO: 116; the V H The region contains the amino acid sequence shown in SEQ ID NO: 3 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% identity with SEQ ID NO: 3; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 4 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% identity with SEQ ID NO: 4; the V HThe region contains the amino acid sequence shown in SEQ ID NO: 3 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% identity with SEQ ID NO: 3; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 117 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% identity with SEQ ID NO: 117; the V H The region contains the amino acid sequence shown in SEQ ID NO: 5 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% identity with SEQ ID NO: 5; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 6 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% identity with SEQ ID NO: 6; the V H The region contains the amino acid sequence shown in SEQ ID NO: 5 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% identity with SEQ ID NO: 5; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 118 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% identity with SEQ ID NO: 118; the V HThe 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% identity with SEQ ID NO: 7; and the V L The region contains 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% identity with SEQ ID NO: 8; the V H The 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% identity with SEQ ID NO: 7; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 119 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% identity with SEQ ID NO: 119; the V H The region contains the amino acid sequence shown in SEQ ID NO: 9 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% identity with SEQ ID NO: 9; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 10 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% identity with SEQ ID NO: 10; the V HThe region contains the amino acid sequence shown in SEQ ID NO: 9 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% identity with SEQ ID NO: 9; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 120 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% identity with SEQ ID NO: 120; the V H The region contains the amino acid sequence shown in SEQ ID NO: 11 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% identity with SEQ ID NO: 11; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 12 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% identity with SEQ ID NO: 12; the V H The region contains the amino acid sequence shown in SEQ ID NO: 11 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% identity with SEQ ID NO: 11; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 121 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% identity with SEQ ID NO: 121; the VH The region contains the amino acid sequence shown in SEQ ID NO: 13 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% identity with SEQ ID NO: 13; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 14 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% identity with SEQ ID NO: 14; or the V H The region contains the amino acid sequence shown in SEQ ID NO: 13 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% identity with SEQ ID NO: 13; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 122 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% identity with SEQ ID NO: 122.

[0025] In some embodiments, the extracellular antigen-binding domain includes V H District and V L Zone, wherein: the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences of SEQ ID NO: 20, 21, and 17, and the V L The region contains amino acid sequences of SEQ ID NO: 25, 26, and 27, respectively; the V H The region contains CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO:35, 36, and 32, and the V LThe region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 40, 41, and 42; the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 50, 51, and 47, and the V L The region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 55, 56, and 57; the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 65, 66, and 62, and the V L The region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 70, 71, and 72; the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively containing the amino acid sequences SEQ ID NO: 80, 81, and 77, and the V L The region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 85, 86, and 87; the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 80, 94, and 91, and the V L The region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 97, 98, and 99; the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 107, 108, and 104, and the V L The region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences of SEQ ID NO: 112, 26, and 113; or the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively containing the amino acid sequences SEQ ID NO: 109, 110, and 111, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 114, 29, and 123.

[0026] In some embodiments, the extracellular antigen-binding domain includes V H District and V L Zone, wherein: the VH The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 1 and 2, or amino acid sequences as shown in SEQ ID NO: 1 and 116, respectively; the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 3 and 4, or amino acid sequences as shown in SEQ ID NO: 3 and 117, respectively; the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO:5 and 6, or amino acid sequences as shown in SEQ ID NO:5 and 118, respectively; the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 7 and 8, or amino acid sequences as shown in SEQ ID NO: 7 and 119, respectively; the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 9 and 10, or amino acid sequences as shown in SEQ ID NO: 9 and 120, respectively; the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 11 and 12, or amino acid sequences as shown in SEQ ID NO: 11 and 121, respectively; or the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 13 and 14, or amino acid sequences as shown in SEQ ID NO: 13 and 122, respectively.

[0027] In some embodiments, the extracellular antigen-binding domain includes V H District and V L Zone, wherein: the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences of SEQ ID NO: 20, 21, and 17, and the V L The region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 25, 26, and 27; the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 35, 36, and 32, and the V LThe region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 40, 41, and 42; the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 65, 66, and 62, and the V L The region comprises CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences SEQ ID NO: 70, 71, and 72; or the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 80, 94, and 91, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 97, 98, and 99.

[0028] In some implementations, the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 1 and 2, or amino acid sequences as shown in SEQ ID NO: 1 and 116, respectively; the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 3 and 4, or amino acid sequences as shown in SEQ ID NO: 3 and 117, respectively; the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 7 and 8, or amino acid sequences as shown in SEQ ID NO: 7 and 119, respectively; or the V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 11 and 12, or amino acid sequences as shown in SEQ ID NO: 11 and 121, respectively.

[0029] In some implementations, the V H The 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% identity with SEQ ID NO: 7; and the V LThe region contains the amino acid sequence shown in SEQ ID NO: 119 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% identity with SEQ ID NO: 119. In some embodiments, the V H The region contains amino acid sequences of SEQ ID NO: 65, 66, and 62, respectively, and the V L The region contains amino acid sequences of SEQ ID NO: 70, 71, and 72, respectively. In some embodiments, the single-chain antibody fragment is or contains a single-chain variable fragment (scFv).

[0030] In some implementations, the V H The area and the V L The regions are connected by flexible connectors. In some embodiments, the connector contains the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 127). In some embodiments, the V... H The area in the V L The carboxyl terminus of the region. In some embodiments, the extracellular antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO: 131, 133, 137, and 141, 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 sequences selected from SEQ ID NO: 137. In some embodiments, the extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 137.

[0031] In some embodiments, the spacer comprises sequences of a hinge region, a CH2 region, and a CH3 region. In some embodiments, the spacer comprises an IgG4 / 2 chimeric hinge region or a modified IgG4 hinge region containing at least one amino acid substitution compared to the human IgG4 hinge; IgG2 / 4 chimeric C H Region 2; and IgG4 C HRegion 3. In some embodiments, the spacer is or comprises (i) the sequence shown in SEQ ID NO: 162; (ii) a functional variant of SEQ ID NO: 162 having 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: 162; or (iii) a consecutive portion of (i) or (ii) having a length of at least 125 amino acids. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 164.

[0032] In some embodiments, the cytoplasmic signaling domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 176 or amino acids 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: 176. In some embodiments, the co-stimulatory signaling region comprises an intracellular signaling domain of CD28, 4-1BB, or ICOS, or a signaling portion thereof. In some embodiments, the co-stimulatory signaling region comprises an intracellular signaling domain of 4-1BB. In some embodiments, the co-stimulatory signal transduction region is or comprises the amino acid sequence shown in SEQ ID NO: 179 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 sequence shown in SEQ ID NO: 179. In some embodiments, the transmembrane domain is or comprises a transmembrane domain derived from CD4, CD28, or CD8. In some embodiments, the transmembrane domain is or comprises a transmembrane domain derived from CD28.

[0033] In some embodiments, the CAR comprises: (1) an extracellular antigen-binding domain that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D), wherein the extracellular antigen-binding domain comprises: (i) a variable heavy chain (V) containing 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. H (ii) a variable light chain (V) containing 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: 119. L (1) region; (2) spacer, said spacer comprising an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; IgG2 / 4 chimeric C H Region 2; and IgG4 C H (3) A transmembrane domain derived from human CD28; and (4) an intracellular signal transduction region comprising a cytoplasmic signal transduction domain of the CD3-ζ (CD3ζ) chain and an intracellular signal transduction domain of 4-1BB.

[0034] In some embodiments, the extracellular antigen-binding domain comprises the V shown in SEQ ID NO: 7. H The amino acid sequence of the region and the V shown in SEQ ID NO: 119 L The extracellular antigen-binding domain comprises the scFv shown in SEQ ID NO: 137. In some embodiments, the transmembrane domain is or comprises the amino acid sequence shown in SEQ ID NO: 173 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: 173.

[0035] In some embodiments, the intracellular signal transduction region comprises (a) a cytoplasmic signal transduction domain of the CD3-ζ (CD3ζ) chain, the cytoplasmic signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 176 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: 176, and (b) an intracellular signal transduction domain of 4-1BB, the intracellular signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 179 or an amino acid sequence having at least or at least about 99% sequence identity with SEQ ID NO: 176. The sequence shown in SEQ ID NO: 179 has an amino acid sequence with 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. In some embodiments, the intracellular signal transduction region is or comprises the sequences shown in SEQ ID NO: 176 and SEQ ID NO: 179.

[0036] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 183 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: 183. In some embodiments, the chimeric antigen receptor is encoded by the nucleotide sequence shown in SEQ ID NO: 182 or a nucleic 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: 182.

[0037] In some implementations, the dose of engineered cells comprises the following number of CAR-expressing T cells: approximately 1.0 x 10⁻⁶. 7 CAR-expressing T cells and 1.2 x 10 9 Between CAR-expressing T cells, at approximately 1.0 x 10 7CAR-expressing T cells with 6.5 x 10 8 Between CAR-expressing T cells, at approximately 1.5 x 10 7 CAR-expressing T cells with 6.5 x 10 8 Between CAR-expressing T cells, at approximately 1.5 x 10 7 CAR-expressing T cells with 6.0 x 10 8 Between CAR-expressing T cells, at approximately 2.5 x 10 7 CAR-expressing T cells with 6.0 x 10 8 Between CAR-expressing T cells, at approximately 5.0 x 10 7 CAR-expressing T cells with 6.0 x 10 8 Between CAR-expressing T cells, at approximately 1.25 x 10 7 CAR-expressing T cells and 1.2 x 10 9 Between CAR-expressing T cells, at approximately 1.5 x 10 7 CAR-expressing T cells and 1.2 x 10 9 Between CAR-expressing T cells, at approximately 5.0 x 10 7 CAR-expressing T cells and 4.5 x 10 8 Between CAR-expressing T cells, or at approximately 1.5 x 10 8 CAR-expressing T cells and 3.0 x 10 8 Between each CAR-expressing T cell, there is an end value.

[0038] In some embodiments, the dose of engineered cells comprises or is about 1.5 x 10⁻⁶. 7 It is approximately 2.5 x 10 7 It is approximately 5.0 x 10 7 It is approximately 7.5 x 10 7 It is approximately 1.5 x 10 8 It is approximately 2.25 x 10 8 , or approximately 3.0 x 10 8 It is approximately 4.5 x 10 8 It is approximately 6.0 x 10 8 It is approximately 8.0 x 10 8 or approximately 1.2 x 10 9 CAR-expressing T cells. In some embodiments, the dose of engineered cells comprises: 7.5 x 10⁻⁶ T cells. 7 One CAR-expressing T cell.

[0039] In some embodiments, the method further includes administering lymphocyte-clearing chemotherapy to the subject prior to the administration of the CAR-T cell therapy. In some embodiments, the lymphocyte-clearing chemotherapy is completed approximately 7 days before the initiation of administration of the dose of the CAR-expressing T cells. In some embodiments, the administration of the lymphocyte-clearing chemotherapy is completed approximately 2 to 7 days before the initiation of administration of the dose of engineered T cells. In some embodiments, the lymphocyte-clearing chemotherapy includes the administration of fludarabine and / or cyclophosphamide. In some embodiments, the lymphocyte-clearing chemotherapy includes the administration of fludarabine and cyclophosphamide.

[0040] In some embodiments, the lymphocyte clearance therapy comprises daily administration of approximately 200-400 mg / m². 2 Cyclophosphamide, containing end-values, can optionally be administered daily at a dose of approximately 300 mg / m². 2 Cyclophosphamide. In some embodiments, the lymphocyte clearance therapy comprises daily administration of approximately 20-40 mg / m². 2 Fludarabine, including the end value, can optionally be administered daily at a dose of approximately 30 mg / m². 2 Fludarabine. In some embodiments, the lymphocyte clearance therapy comprises administering fludarabine and cyclophosphamide for 2–4 days, optionally 3 days.

[0041] In some embodiments, the lymphocyte clearance therapy includes administration of bendamustine. In some embodiments, the lymphocyte clearance therapy includes daily administration of approximately 50-130 mg / m². 2 Bendamustine, containing end-point, can optionally be administered daily at a dose of approximately 90 mg / m². 2 Bendamustine. In some embodiments, the lymphocyte clearance therapy includes administration of bendamustine for 1-3 days, optionally 2 days.

[0042] In some implementations, the method further includes administering bridging therapy to the subject after leukapheresis and before lymphocyte clearance chemotherapy.

[0043] In some embodiments, the subject has received one or more prior therapies. In some embodiments, the subject has received at least one but no more than three prior therapies. In some embodiments, the subject has received at least three prior therapies. In some embodiments, the prior therapy is a proteasome inhibitor, an immunomodulatory agent, an anti-CD38 antibody, a prior therapy including autologous hematopoietic stem cell transplantation (HSCT), or a combination of any of the foregoing.

[0044] In some embodiments, the dosing regimen of the bispecific antibody in combination with the CAR-T cell therapy ensures that the subject does not develop toxicities characterized by one or more of the following: (i) any grade 4 CRS and CRS that does not resolve to < grade 2 within 72 hours; (ii) any duration of grade 3 ICANS or neurotoxicity (NT); (iii) any duration of grade higher toxicity involving vital organs (e.g., heart, lungs); (iv) all other grade 3 toxicities that do not resolve to ≤ grade 2 within 72 hours and are not attributable to underlying disease or lymphocyte clearance chemotherapy, excluding non-CRS hepatic toxicities, wherein liver transaminase levels are ≥ 3x the upper limit of normal (ULN), accompanied by an increase in total bilirubin to > 2x. ULN, but without an initial finding of cholestasis (e.g., elevated serum alkaline phosphatase), and no other cause can be found to explain the simultaneous elevation of transaminases and total bilirubin, such as hepatitis A, B, or C, pre-existing liver disease or acute liver disease, or another drug that could cause the observed liver injury; (v) Grade 3 aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) elevations lasting ≤ 14 days or Grade 4 AST and / or ALT elevations lasting ≤ 7 days; (vi) Isolated Grade 3 or 4 asymptomatic laboratory electrolyte abnormalities not directly related to major organ toxicity that occur during certain treatments and resolve to ≤ Grade 2 within ≤ 7 days with or without intervention; (vii) Grade 4 thrombocytopenia that does not resolve to Grade 3 or lower within 14 days or is accompanied by clinically significant bleeding, or Grade 3 thrombocytopenia accompanied by clinically significant bleeding; (viii) Despite the use of granulocyte colony-stimulating factor (G-CSF), the patient experienced a grade 4 neutropenia that did not resolve to grade 3 or lower within 14 days; (ix) any grade 5 toxicity unrelated to disease progression; (x) any macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH); or (xi) any thrombotic event of grade 3 or higher.

[0045] In some implementations, the subjects do not develop serious toxicity.

[0046] In some of the provided embodiments, the subject is a human being. In some embodiments, the subject is an adult subject. In some embodiments, the subject is 18 years of age or older. Attached Figure Description

[0047] Figure 1 An exemplary treatment regimen including anti-GPRC5D CAR T-cell therapy and anoxtabimab administration is described. Detailed Implementation

[0048] The provided embodiments include combination therapies involving the administration of T-cell therapy (e.g., CAR-expressing T cells) and the administration of combination agents (e.g., bispecific antibodies, such as T-cell connectors targeting BCMA). This document provides combination therapies for treating subjects with cancer, said combination therapies involving the administration of T-cell therapy (e.g., CAR-T cells) and additional agents (e.g., bispecific antibodies; such as T-cell connectors targeting BCMA) for the treatment of subjects with cancer or tumors (such as multiple myeloma). In some embodiments, the methods provided herein are based on the administration of a combination of CAR T-cell therapy targeting GPRC5D and a combination agent (including bispecific antibodies, such as T-cell connectors targeting BCMA), wherein the CAR contains an scFv antigen-binding domain targeting GPRC5D. Therefore, in some embodiments, the methods and uses include administering to a subject in adoptive cell therapy a combination of T cells expressing a genetically engineered (recombinant) cell surface receptor and a bispecific antibody (such as a T cell connector against BCMA), wherein the genetically engineered (recombinant) cell surface receptor is typically a chimeric receptor (such as a chimeric antigen receptor (CAR)) that recognizes an antigen expressed by, associated with, and / or specific to the cell type from which it originates.

[0049] In some implementations, the T-cell adaptor targeting BCMA binds to both BCMA and CD3.

[0050] This article also provides methods for treating relapsed and / or refractory multiple myeloma (RRMM). In some embodiments, the method includes administering CAR-T cell therapy to a subject comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D), followed by administration of a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3. In some embodiments, the bispecific antibody is administered no earlier than 60 days after administration of CAR T cell therapy.

[0051] In some embodiments, the bispecific antibody is a T-cell adaptor targeting BCMA. In some embodiments, the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody.

[0052] In some implementations, combination therapies involving the administration of CAR-T cells and bispecific antibodies (e.g., T-cell adjuvants targeting BCMA) can produce improved anti-myeloma activity through at least two mechanisms. In some cases, the improved activity can synergize with each individual therapy. First, given the heterogeneity of GPRC5D and BCMA target expression in myeloma cells has been demonstrated in individual patients, combination anti-GPRC5D and anti-BCMA targeted therapies can produce greater depth of tumor clearance, increased clearance of minimal residual disease (MRD), and potentially greater durability of response. Second, administration of bispecific antibodies (e.g., T-cell adjuvants targeting BCMA) following CAR-T cell therapy is expected to redirect endogenous T cells to kill BCMA-expressing tumor cells. Bispecific antibodies (e.g., T-cell adjuvants targeting BCMA) can also bind to residual CAR T cells, thereby allowing dual targeting of BCMA via bispecific antibodies (e.g., T-cell adjuvants targeting BCMA) and GPRC5D via CAR T cells, prolonging combined endogenous T-cell and CAR T-cell immune surveillance against the tumor.

[0053] Multiple myeloma (MM) is a hematologic malignancy characterized by the uncontrolled proliferation of monoclonal plasma cells in the bone marrow, leading to excessive production of monoclonal immunoglobulins and immunosuppression (Al-Hujaily 2016; Dimopoulos, 2015). Adoptive T-cell therapies (such as CAR-T cell therapy) have shown promise for treating multiple myeloma, with clinical efforts primarily focused on targeting B-cell maturation antigen (BCMA). However, although BCMA is expressed on many malignant plasma cells, its expression levels can be heterogeneous in some cases. In some respects, heterogeneity in target antigen expression can lead to variable or inconsistent responses. In other respects, changes in BCMA expression on the cell surface over time have also been observed due to the shedding of extracellular domains mediated by γ-secretase. Similar to observations regarding CD19 and CD22 CAR antigens, BCMA antigen downregulation has been reported in patients with relapsed multiple myeloma (MM) following BCMA-targeted T-cell therapy (Brudno et al. (2018) J. Clin. Oncol., JCO2018778084; Cohen et al. (2017) Blood 130:505). Furthermore, in some contexts, recombinant receptors can exhibit antigen-independent activity or signaling (also known as “tetanic signaling”), which can lead to undesirable effects such as increased differentiation and / or exhaustion of T cells expressing the recombinant receptor. In some respects, such activity may limit the activity, function, or potency of T cells. In some cases, during the engineering and in vitro expansion of cells for recombinant receptor expression, said cells may exhibit exhaustion-indicating phenotypes due to tetanic signaling via the recombinant receptor. In some cases, alternative or additional T-cell therapy approaches targeting MM are needed.

[0054] The provided implementation scheme relates to GPRC5D as a CAR T-cell target for multiple myeloma. GPRC5D (Uniprot accession number Q9NZD1, e.g., SEQ ID NO: 207) is a class C5 family member D of G protein-coupled receptors, belonging to the RAIG (retinoic acid-inducible gene-1) family. It is a seven-transmembrane helical 39 kDa G protein-coupled receptor with two reported isoforms, the difference of which occurs at the intracellular C-terminus of the protein. GPRC5D is expressed at high levels in multiple myeloma and, overall, at low levels in most normal tissues.

[0055] In some embodiments, the method includes administering the combination therapy to a selected or identified subject with a certain prognosis or risk of multiple myeloma. In some embodiments, the method, use, and article involve or are used to treat a subject, involving the selection or identification of a specific group or subset of subjects, such as any subject group as described, based on a specific disease type, diagnostic criteria, prior treatment, and / or response to prior treatment. In some embodiments, the method involves treating a subject who has relapsed after remission following treatment with two or more prior therapies or who has become refractory to one or more prior therapies. In some embodiments, the subject has relapsed or is refractory (R / R) to one or more prior therapies (e.g., one or more lines of standard therapy). In some embodiments, the method involves treating a subject who has relapsed after remission following treatment with two or more prior therapies or who has become refractory to two or more prior therapies. In some embodiments, the subject has relapsed or is refractory (R / R) to two or more prior therapies (e.g., two or more lines of standard therapy). In some embodiments, the method involves treating a subject who has relapsed after remission following treatment with three or more prior therapies or who has become refractory to three or more prior therapies. In some implementations, the subject has relapsed or is refractory to three or more prior therapies (e.g., three or more lines of standard therapy).

[0056] In some respects, the provided methods, compositions, uses, and articles achieve improved and superior responses to available therapies, including T-cell therapy alone or combination agents alone (e.g., bispecific antibodies). In some embodiments, the improved or superior response is compared to current standard of care (SOC).

[0057] Also provided are combinations and articles (such as kits) comprising compositions containing T-cell therapy and / or compositions containing combination agents (e.g., bispecific antibodies); and the use of such compositions and combinations for the treatment or prevention of cancer (e.g., multiple myeloma). In some embodiments, the cancer is cancer expressing GPRC5D. In some embodiments, the cancer is cancer expressing BCMA. In some embodiments, the cancer is associated with the expression of both GPRC5D and BCMA.

[0058] All publications (including patent documents, scientific articles, and databases) mentioned in this application are incorporated herein by reference in their entirety for all purposes, to the extent that each individual publication is incorporated individually by reference. Where the definitions described herein are contrary to or otherwise inconsistent with those described in patents, applications, published applications, and other publications incorporated herein by reference, the definitions described herein shall prevail over those incorporated herein by reference.

[0059] The chapter titles used in this article are for organizational purposes only and should not be construed as limiting the topics described. I. Combination Therapy

[0060] This document provides methods of combination therapy, the methods comprising the administration and use (e.g., for therapeutic and prophylactic purposes) of a combination agent comprising a GPRC5D-binding recombinant receptor (e.g., a CAR) and / or a composition comprising therein. In some embodiments, the GPRC5D-binding recombinant receptor is an anti-GPRC5D CAR administered to a subject in combination with a combination agent. In some embodiments, the combination agent is a bispecific antibody. In some embodiments, the bispecific antibody is a T-cell connective targeting BCMA. In some embodiments, the T-cell connective targeting BCMA binds to both BCMA and CD3.

[0061] This article also provides methods for treating relapsed and / or refractory multiple myeloma (RRMM). In some embodiments, the method includes administering a combination therapy, wherein the combination therapy comprises CAR-T cell therapy and a bispecific antibody (e.g., a T-cell connective targeting BCMA). In some embodiments, the method includes administering to a subject CAR-T cell therapy comprising a dose of engineered cells containing T cells expressing a CAR that specifically binds to human GPRC5D, followed by administration of a bispecific antibody that binds to BCMA and CD3. In some embodiments, the bispecific antibody is administered no earlier than 60 days after administration of CAR T-cell therapy. In some embodiments, the bispecific antibody is a T-cell connective targeting BCMA. In some embodiments, the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody.

[0062] In some embodiments, engineered cells expressing the GPRC5D-binding molecule (e.g., CAR) and / or the molecule (e.g., recombinant receptor) described herein are administered as part of a combination therapy or combination treatment, for example, simultaneously, sequentially, or intermittently, with one or more additional therapeutic interventions involving the administration of a T-cell connector targeting BCMA. Methods of combination therapy or combination treatments are also provided, comprising the GPRC5D-binding recombinant receptor (e.g., CAR), cells, and / or compositions described herein, and one or more additional therapeutic interventions involving the administration of a T-cell connector targeting BCMA.

[0063] The provided embodiments include methods of administration and uses comprising a combination of GPRC5D-targeted cell therapy and a bispecific antibody. Such methods and uses include, for example, treatment methods and uses involving administering the combination to a subject suffering from a GPRC5D-related disease, condition, or disorder, such as a disease, condition, or disorder associated with GPRC5D expression and / or in which cells or tissues express (e.g., specifically express) GPRC5D. In some embodiments, the combination is administered in an effective amount to treat the disease or disorder. In some embodiments, the method is performed by administering the combination to a subject who has, already has, or is suspected of having a disease or condition. In some embodiments, the method thereby treats the subject's disease, condition, or disorder. The use of the combination for treating GPRC5D-related diseases or disorders is also provided herein, such as in a treatment regimen.

[0064] This article also provides the use of anti-GPRC5D CAR cell therapy (e.g., T-cell therapy) and bispecific antibodies as a combination therapy in such methods and treatments for subjects with diseases or disabilities, and in the preparation of pharmaceutical agents to implement such treatments. This article also provides the use of anti-GPRC5D CAR cell therapy (e.g., T-cell therapy) in combination with bispecific antibodies in such methods and treatments for subjects with diseases or disabilities, and in the preparation of pharmaceutical agents to implement such treatments in combination with bispecific antibodies. This article also provides the use of bispecific antibodies in combination with anti-GPRC5D CAR cell therapy (e.g., T-cell therapy) in such methods and treatments for subjects with diseases or disabilities, and in the preparation of pharmaceutical agents to implement such treatments in combination with anti-GPRC5D CAR cell therapy (e.g., T-cell therapy).

[0065] As used herein, “treatment” (and its grammatical variations such as “treat” or “treating”) means the complete or partial improvement or relief of a disease or symptom or disorder, or related symptoms, adverse events, effects or outcomes, or phenotype. Desired therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction of the rate of disease progression, improvement or relief of the disease state, and relief or improvement of prognosis. The term does not imply a complete cure of the disease or the complete elimination of any symptoms or one or more effects on all symptoms or outcomes.

[0066] As used herein, “adverse event” means any new unfortunate medical event or exacerbation of a pre-existing medical condition that occurs in a clinical study participant after the informed consent has been given, whether or not it is considered related to the study intervention. Therefore, an adverse event can be any adverse or unexpected sign (such as abnormal laboratory test results), symptom, or illness that is temporarily related to the study intervention.

[0067] As used in this article, “delaying disease development” means postponing, hindering, slowing, delaying, stabilizing, inhibiting, and / or postponing the development of a disease (such as cancer). This delay can vary in length, depending on the patient’s medical history and / or the subject being treated. Sufficient or significant delay can effectively cover prevention, as the subject will not develop the disease. For example, it may delay the development of advanced cancer, such as metastasis.

[0068] As used herein, “prevention” includes providing prevention of the onset or recurrence of a disease in a subject who may be susceptible to the disease but has not yet been diagnosed with it. In some embodiments, the provided molecules and compositions are used to delay the development of a disease or slow its progression.

[0069] As used herein, "inhibitory" function or activity is a reduction of function or activity when compared to conditions that are identical except for the target condition or parameter, or alternatively, when compared to another condition. For example, an antibody, composition, or cell that inhibits tumor growth reduces the tumor growth rate compared to the tumor growth rate in the absence of the antibody, composition, or cell.

[0070] The term "pharmaceutical formulation" refers to a preparation which is in a form that enables the bioactivity of the active ingredient contained therein to be effective and does not contain any additional components that would have unacceptable toxicity to a subject administering the formulation.

[0071] "Pharmaceutically acceptable carriers" refer to components in a drug formulation that are non-toxic to the subject, except for the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffer solutions, excipients, stabilizers, or preservatives.

[0072] In the context of administration, the “effective amount” of a drug (e.g., a pharmaceutical formulation, a conjugating molecule, an antibody, a cell, or a composition) refers to the amount that effectively achieves the desired outcome (such as a therapeutic or preventative outcome) at the necessary dose / amount and for the necessary time period.

[0073] A “therapeutic effective amount” of a pharmaceutical agent (such as a pharmaceutical formulation, conjugated molecule, antibody, cell, or composition) refers to the amount that effectively achieves the desired therapeutic outcome (such as for the treatment of a disease, condition, or disorder) and / or the therapeutic pharmacokinetic or pharmacodynamic effect at the necessary dose and for the necessary time period. Therapeutic effective amounts can vary depending on factors such as disease state, subject age, sex, and weight, and the cell population administered. In some embodiments, the provided methods involve administering the molecule, antibody, cell, and / or composition at an effective amount (e.g., a therapeutic effective amount).

[0074] "Effective preventive dose" refers to the amount that effectively achieves the desired preventive outcome at the necessary dose and within the necessary time period. It is usually, but not always, less effective because the preventive dose is administered to the subject before or at an earlier stage of the disease.

[0075] As used herein, "subject" or "individual" refers to a mammal. In some embodiments, "mammal" includes humans, non-human primates, livestock and farm animals, as well as zoo, sporting, or pet animals such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject is a human.

[0076] The diseases to be treated include any disease or disorder associated with GPRC5D or that specifically expresses GPRC5D and / or that have been targeted for treatment with GPRC5D (which may also be referred to interchangeably herein as "GPRC5D-related diseases or disorders"). Cancers associated with GPRC5D expression include hematologic malignancies such as myeloma, for example, multiple myeloma. In some embodiments, the GPRC5D-related disease or disorder is a B-cell-related disorder or malignancy. In some embodiments, the GPRC5D-related disease or disorder is multiple myeloma or Waldenström macroglobulinemia. In some embodiments, the disease or disorder is multiple myeloma. The provided embodiments include cells, populations, and compositions for administration, for example, via adoptive cell therapy (such as adoptive T-cell therapy) to a subject suffering from relapsed and / or refractory multiple myeloma (RRMM).

[0077] In some embodiments, the method can identify subjects who have, are suspected of having, or are at risk of having a disease or disorder related to GPRC5D. Therefore, a method is provided for identifying subjects with a disease or disorder related to GPRC5D expression and selecting them for treatment with the provided combination of GPRC5D-targeted cell therapy and bispecific antibody.

[0078] For example, subjects can be screened for the presence of diseases or disorders associated with elevated GPRC5D expression, such as cancers expressing GPRC5D. In some embodiments, the method includes screening for or detecting the presence of GPRC5D-related diseases (e.g., tumors). Thus, in some aspects, samples can be obtained from patients suspected of having diseases or disorders associated with elevated GPRC5D expression, and the expression level of GPRC5D can be determined. In some aspects, subjects who test positive for GPRC5D-related diseases or disorders can be selected for treatment using the method of the present invention, and a therapeutically effective amount of a combination of GPRC5D-targeting cell therapy and a bispecific antibody can be administered.

[0079] In some embodiments, the subject has a history of relapsed / refractory multiple myeloma. In some embodiments, the subject has refractory or relapsed disease, for example, after treatment with BCMA-specific antibodies and / or cells and / or other therapies (including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT) (e.g., allogeneic HSCT or autologous HSCT)). In some embodiments, the administration is still effective in treating the subject even though the subject has developed resistance to another GPRC5D-targeted therapy. In some embodiments, the subject has not yet relapsed but is identified as being at risk of relapse, e.g., at high risk of relapse, and therefore a compound or composition is administered prophylactically, for example, to reduce the likelihood of relapse or to prevent relapse.

[0080] In some implementations, the subject is eligible for transplantation, such as a hematopoietic stem cell transplant (HSCT) (e.g., allogeneic HSCT or autologous HSCT). In some such implementations, although the subject is eligible for transplantation, they have not previously received a transplant before administration of the combination of GPRC5D-targeted cell therapy and a bispecific antibody.

[0081] In some implementations, the subject is an ineligible recipient, such as a ineligible recipient of hematopoietic stem cell transplantation (HSCT) (e.g., allogeneic HSCT or autologous HSCT). In some such implementations, such subjects are administered a combination of GPRC5D targeted cell therapy and a bispecific antibody according to the implementation scheme provided herein.

[0082] In some embodiments, the subject has received one or more prior therapies before initiating administration of the combination comprising GPRC5D targeted cell therapy and a bispecific antibody. In some embodiments, the subject has received at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more prior therapies. In some embodiments, the subject has received at least 1, 2, 3, 4, or more prior therapies. In some embodiments, the subject has received at least 3 prior therapies. In some embodiments, the subject has received at least 2 prior therapies. In some embodiments, the subject has received at least 1 prior therapy. In some embodiments, the subject has received at least one but no more than three prior therapies. In some embodiments, prior therapies include, but are not limited to, immunomodulatory agents (e.g., thalidomide, lenalidomide, pomalidomide), proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib), dexamethasone, anti-CD38 antibodies, and autologous hematopoietic stem cell transplantation (HSCT). In some embodiments, induction with or without HSCT and with or without maintenance therapy are considered as one regimen. In some implementations, prior therapy includes prior therapy targeting BCMA and / or GPRC5D.

[0083] In some aspects, the subject has relapsed after one or more of the aforementioned one or more prior therapies (e.g., each alone), or is already refractory to one or more of the aforementioned one or more prior therapies (e.g., each alone). In some embodiments, the subject has undergone at least two consecutive treatment cycles for each regimen unless disease progression is the best response to the regimen.

[0084] In some implementations, the subject has undergone at least one complete treatment cycle unless disease progression is the best response to the treatment regimen. In some implementations, the subject has been treated with at least one, but no more than three, prior antimyeloma treatment regimens, including proteasome inhibitors and immunomodulatory agents.

[0085] In some embodiments, the methods described herein can be used to treat diseases, conditions, and disorders in which GPRC5D is expressed. In some embodiments, the disease or disorder associated with GPRC5D is a B-cell-related disorder or malignancy. In some embodiments, the disease or disorder associated with GPRC5D is multiple myeloma or Waldenström macroglobulinemia. In some embodiments, the disease or disorder is multiple myeloma. In some embodiments, the subject treated with the methods is diagnosed with multiple myeloma with relapsed and / or refractory disease.

[0086] In some embodiments, the method may involve excluding specific subjects from receiving therapy using the provided anti-GPRC5D antibody, recombinant receptor, and / or cells containing such receptor, based on specific criteria, diagnoses, or indications. In some embodiments, the subject does not have active plasma cell leukemia (PCL) or a history of PCL at the time of administration of the combination.

[0087] In some implementations, the treatment does not induce an immune response in the subject to the therapy, and / or induces such a response to an extent that is insufficient to effectively stop the disease or condition. In some respects, the degree of immunogenicity and / or graft-versus-host reaction is less than that observed with different but comparable treatments.

[0088] In some implementations, the disease or disorder to be treated is multiple myeloma. In some implementations, measurable disease criteria for multiple myeloma may include: (1) serum M protein 1 g / dL or higher; (2) urinary M protein 200 mg or higher / 24 hours; (3) serum free light chain (sFLC) levels of 10 mg / dL or higher with an abnormal κ to λ ratio. In some cases, light chain disease is only applicable to subjects without measurable disease in serum or urine.

[0089] In some implementations, the Eastern Cooperative Oncology Group (ECOG) performance status indicator can be used to assess or select subjects for treatment, such as those with poor performance due to prior therapy (see, for example, Oken et al. (1982) Am J Clin Oncol. 5:649-655). The ECOG performance status scale describes a patient’s functional level in terms of self-care, daily activities, and physical abilities (e.g., walking, working, etc.). In some implementations, an ECOG performance status of 0 indicates that the subject can perform normal activities. In some respects, a subject with an ECOG performance status of 1 exhibits some limitations in physical activity, but the subject can walk fully. In some respects, a patient with an ECOG performance status of 2 can walk more than 50%. In some cases, a subject with an ECOG performance status of 2 may also be able to care for themselves; see, for example, Sørensen et al. (1993) Br J Cancer 67(4) 773-775. In some implementations, subjects to whom the methods or treatments provided herein are administered include those with an ECOG performance status of 0 or 1.

[0090] In some implementations, the administration can treat the subject even though the subject has developed resistance to another therapy.

[0091] In some embodiments, when administered to subjects according to the embodiments described herein, the combination is able to achieve an objective response (OR) in at least 50%, 60%, 70%, 80%, 90%, or 95% of the subjects administered. In some embodiments, OR includes subjects achieving a strict complete response (sCR), a complete response (CR), a very good partial response (VGPR), a partial response (PR), and a minimum response (MR). In some embodiments, when administered to subjects according to the embodiments described herein, the combination is able to achieve a strict complete response (sCR), a complete response (CR), a very good partial response (VGPR), or a partial response (PR) in at least 50%, 60%, 70%, 80%, or 85% of the subjects administered. In some embodiments, when administered to subjects according to the embodiments described herein, the combination is able to achieve a strict complete response (sCR) or a complete response (CR) in at least 20%, 30%, 40%, 50%, 60%, or 70% of the subjects administered.

[0092] In some implementations, for example, treatment according to the provided method may result in a lower rate and / or lower degree of toxicity, toxic outcome or symptoms, toxicity-promoting features, factors or characteristics, such as symptoms or outcomes associated with or indicating cytokine release syndrome (CRS) or neurotoxicity (such as severe CRS or severe neurotoxicity), compared to the administration of other therapies. A.GPRC5D Targeted Cell Therapy

[0093] In some respects, GPRC5D binding agents (such as recombinant receptors or chimeric antigen receptors that bind to the GPRC5D molecule) and polynucleotides encoding GPRC5D-binding cell surface proteins (such as recombinant receptors (e.g., CARs)) and cells expressing such receptors are provided. GPRC5D-binding cell surface proteins typically contain antibodies (e.g., antigen-binding antibody fragments) and / or other binding peptides that specifically bind to GPRC5D (such as GPRC5D proteins, such as human GPRC5D protein). In some respects, the agent binds to the extracellular portion of GPRC5D.

[0094] The provided polynucleotides include those encoding recombinant receptors (such as antigen receptors) that specifically bind to GPRC5D. In some aspects, encoded receptors (such as those containing GPRC5D-binding peptides) and their compositions and articles, as well as uses, are also provided.

[0095] GPRC5D binding peptides include antibodies, such as single-chain antibodies (e.g., antigen-binding antibody fragments) or portions thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor, such as those containing an anti-GPRC5D antibody or its antigen-binding fragment. The provided polynucleotide can be incorporated into a construct, such as a deoxyribonucleic acid (DNA) or RNA construct, such as those that can be introduced into cells to express the recombinant GPRC5D binding receptor. 1. GPRC5D chimeric antigen receptor

[0096] The provided GPRC5D binding receptors typically contain both extracellular binding molecules and intracellular signaling domains. These receptors include antibody-containing peptides, such as recombinant cell surface receptors containing anti-GPRC5D antibodies. Such receptors also include chimeric antigen receptors containing such antibodies.

[0097] The recombinant receptors provided include antigen receptors (including GPRC5D binding fragments). Recombinant receptors include antigen receptors that specifically bind to GPRC5D, such as antigen receptors containing anti-GPRC5D antibodies (e.g., GPRC5D antigen-binding fragments). Antigen receptors include functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). Cells expressing the recombinant receptors and their use in adoptive cell therapies, such as the treatment of diseases and disorders associated with GPRC5D expression (e.g., multiple myeloma), are also provided.

[0098] In some embodiments, the CAR includes an extracellular antigen-binding domain that specifically binds to GPRC5D, a spacer sequence between the extracellular antigen-binding domain and the transmembrane domain, a transmembrane domain, and an intracellular signaling region comprising a cytoplasmic signaling domain containing a CD3-ζ (CD3ζ) chain and a co-stimulatory signaling region. In some embodiments, the spacer sequence is a sequence containing a hinge domain. In some embodiments, the spacer sequence is an immunoglobulin hinge spacer with a length of at least 125 amino acids. a. Extracellular antigen-binding domain

[0099] Chimeric receptors include chimeric antigen receptors (CARs). Chimeric receptors (such as CARs) typically contain an extracellular antigen-binding domain that includes, is, or contains an anti-GPRC5D antibody (such as an antibody fragment). Therefore, a chimeric receptor (e.g., a CAR) typically contains one or more GPRC5D binding molecules, such as one or more antigen-binding fragments, domains, or portions, or one or more antibody variable regions and / or antibody molecules (as described herein), in its extracellular portion.

[0100] The term "antibody" is used in the broadest sense in this article and includes both polyclonal and monoclonal antibodies, including complete antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, and heavy chain variable (V) fragments capable of specifically binding antigens. H This term encompasses single-chain antibody fragments (including single-chain variable fragments (scFv)) and single-domain antibody fragments (e.g., sdAb, sdFv, nanobodies). The term covers genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide bodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugated antibodies, multispecific (e.g., bispecific or trispecific) antibodies, biantibodies, triantibodies and tetraantibodies, tandem biscFvs, and tandem triscFvs. Unless otherwise stated, the term "antibody" should be understood to encompass the functional antibody fragment, also referred to herein as an "antigen-binding fragment." The term also covers complete or full-length antibodies, including antibodies of any class or subclass (including IgG and its subclasses, IgM, IgE, IgA, and IgD).

[0101] The terms “complementarity-determining region” and “CDR”, synonymous with “hypervariant region” or “HVR”, are known in the art to refer to a discontinuous amino acid sequence within the variable region of an antibody that confers antigen specificity and / or binding affinity. Typically, 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. The terms “frame region” and “FR” are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, 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.

[0102] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many well-known schemes, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0103] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structure alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the most common antibody region sequences, where insertions are provided by insert letters (e.g., "30a"), and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways. The AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the scheme used by Oxford Molecular's AbM antibody modeling software.

[0104] Table 1 below lists exemplary positional boundaries of CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and 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. FRs are located between CDRs; for example, FR-L1 precedes CDR-L1, FR-L2 is between CDR-L1 and CDR-L2, FR-L3 is between CDR-L2 and CDR-L3, and so on. Note that because the Kabat numbering scheme shown places insertions at H35A and H35B, when numbering using the Kabat numbering convention shown, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the loop length.

[0105] Therefore, unless otherwise specified, the “CDR” or “complementarity-determining region” or individually designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or its region (such as its variable region) should be understood to encompass a (or specific) complementarity-determining region as defined by any of the foregoing schemes or other known schemes. For example, in a statement that a particular CDR (e.g., CDR-H3) contains a given V H or V LIn the case of the amino acid sequence of the corresponding CDR in the variable region amino acid sequence, it should be 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, a specific CDR sequence is specified. Exemplary CDR sequences of the provided antibody are described using various numbering schemes, but it should be understood that the provided antibody may include CDRs as described according to any other numbering scheme described above or other numbering schemes known to a skilled person.

[0106] Similarly, unless otherwise specified, a given antibody or its regions, such as the FRs of its variable region or one or more individually designated FRs (e.g., FR-H1, FR-H2, FR-H3, FR-H4), should be understood to encompass one (or a specific) frame region as defined by any known protocol. In some cases, an identification protocol for identifying a particular CDR, FR, or multiple specific FRs or CDRs is specified, such as a CDR defined by the Kabat, Chothia, AbM, or Contact methods or other known protocols. In other cases, the specific amino acid sequence of the CDR or FR is given.

[0107] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that participates in the binding of the antibody to the antigen. The variable regions of the heavy and light chains of natural antibodies (V1 and V2, respectively) H and V L They typically have similar structures, with each domain containing four conserved frame regions (FRs) and three core regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007). Single V H or V L The structural domain may be sufficient to confer antigen-binding specificity. Furthermore, V-terminal molecules derived from antibodies that bind to specific antigens can be used. H or V L The domains are separated to isolate antibodies that bind to the antigen, in order to screen for complementary V antibodies. H or V L Library of structural domains (see, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0108] The antibodies included in the provided CAR include antibody fragments. An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than the complete antibody that contains a portion of the antigen bound to the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; linear antibodies; and heavy chain variable (V) antibodies. H ) region, single-chain antibody molecules (such as scFv) and those containing only V H Single-domain antibodies in the region; and multispecific antibodies formed from antibody fragments. In some embodiments, the antigen-binding domain in the provided CAR is or includes a variable heavy chain (V... H ) region and variable light chain (V L Antibody fragments containing the heavy chain variable (V) region. In a particular embodiment, the antibody is an antibody containing the heavy chain variable (V) region. H ) region and / or light chain variable (V L Single-chain antibody fragments (e.g., scFv) in the region )

[0109] A single-domain antibody (sdAb) is an antibody fragment containing all or part of the heavy chain variable region or all or part of the light chain variable region of an antibody. In some embodiments, the single-domain antibody is a human single-domain antibody.

[0110] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and production from recombinant host cells. In some embodiments, the antibody is a recombinantly generated fragment, such as a fragment containing an arrangement not naturally occurring (e.g., those having two or more antibody regions or chains linked by synthetic linkers (e.g., peptide linkers), and / or a fragment that can be generated without enzymatic digestion of naturally occurring intact antibodies. In some aspects, the antibody fragment is an scFv.

[0111] A “humanized” antibody is an antibody in which all or substantially all of the CDR amino acid residues are derived from a non-human CDR and all or substantially all of the FR amino acid residues are derived from a human FR. A humanized antibody may optionally include at least a portion of the 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 undergone humanization to generally reduce its immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced by corresponding residues from a non-human antibody (e.g., an antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0112] The anti-GPRC5D antibody (such as an antibody fragment) included in the provided CAR includes human antibodies. "Human antibody" is an antibody having an amino acid sequence corresponding to an amino acid sequence produced by a human or human cell or a non-human source utilizing a human antibody library or other human antibody-encoding sequences (including human antibody libraries). The term does not include humanized forms of non-human antibodies containing non-human antigen-binding regions, such as those for which all or substantially all CDRs are non-human CDRs. The term includes antigen-binding fragments of human antibodies.

[0113] The antibodies included in the provided CAR include those that are monoclonal antibodies, including monoclonal antibody fragments. As used herein, the term "monoclonal antibody" refers to an antibody obtained from or within a substantially homogeneous group of antibodies (i.e., the individual antibodies constituting the group are identical, except for those containing naturally occurring mutations or possible variants generated during the production of the monoclonal antibody formulation, which are typically present in small amounts). In contrast to polyclonal antibody formulations, which typically include different antibodies targeting different epitopes, each monoclonal antibody in a monoclonal antibody formulation targets a single epitope on the antigen. The term should not be construed as requiring the antibody to be produced by any particular method. Monoclonal antibodies can be prepared using a variety of techniques, including but not limited to hybridoma generation, recombinant DNA methods, phage display, and other antibody display methods.

[0114] In some implementations, the CAR comprises one or more GPRC5D binding moieties of the antibody molecule, such as the variable heavy chain (V) of the antibody. H ) region and / or light chain variable (V LThe GPRC5 binding CAR contains an antibody (e.g., an scFv antibody fragment) that imparts GPRC5 binding properties to the provided CAR. In some embodiments, the provided GPRC5 binding CAR contains an antibody (e.g., an anti-GPRC5 antibody) or an antigen-binding fragment thereof that confers GPRC5 binding properties to the provided CAR. In some embodiments, the antibody or antigen-binding domain may be any of the described anti-GPRC5D antibodies or derived from any of the described anti-GPRC5D antibodies (see, for example, WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245, WO 2023 / 134718). Any such anti-GPRC5D antibody or antigen-binding fragment may be used in the provided CAR. In some embodiments, the anti-GPRC5DCAR contains an antigen-binding domain comprising a variable heavy chain (V1) of an antibody derived from the antibodies described in WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245 or WO 2023 / 134718. H ) region and / or variable light chain (V L )district.

[0115] In some embodiments, the antibody (e.g., an anti-GPRC5D antibody or an antigen-binding fragment) contains a variable (V) heavy chain and / or light chain as described above. H or V L The region or sufficient antigen-binding portion thereof. In some embodiments, the anti-GPRC5D antibody (e.g., the antigen-binding fragment) comprises V containing CDR-H1, CDR-H2 and / or CDR-H3 as described above. H The region sequence or a sufficient antigen-binding portion thereof. In some embodiments, the anti-GPRC5D antibody (e.g., an antigen-binding fragment) comprises V containing CDR-L1, CDR-L2 and / or CDR-L3 as described above. L The region sequence or sufficient antigen-binding fragment. In some embodiments, the anti-GPRC5D antibody (e.g., an antigen-binding fragment) comprises V containing CDR-H1, CDR-H2, and / or CDR-H3 as described above. H The region sequence; and includes V containing CDR-L1, CDR-L2 and / or CDR-L3 as described above. LThe antibody also includes those 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 or at least about 99% identical to such a sequence.

[0116] In some embodiments, the antibody or antibody fragment in the provided CAR has a V of any antibody or antibody-binding fragment described in any one of WO 2016 / 090312, WO2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245 and WO2023 / 134718. H district.

[0117] In some embodiments, the CAR contains an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment having a heavy chain variable (V) H The heavy chain variable region has an amino acid sequence selected from any one of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13, or a V sequence selected from any one of SEQ ID NO: 1, 3, 5, 7, 9, 11 or 13. H The amino acids in the region have an amino acid sequence with 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; or contain amino acid sequences present in such a region. H The CDR-H1, CDR-H2 and / or CDR-H3 in the sequence.

[0118] In some implementations, the V of the antibody or its antigen-binding fragment H The region contains CDR-H1, CDR-H2, and / or CDR-H3 according to Kabat numbers. In some embodiments, the V of the antibody or its antigen-binding fragment... H The region includes CDR-H1, CDR-H2, and / or CDR-H3 according to Chothia designations. In some embodiments, the V of the antibody or its antigen-binding fragment... H The region contains CDR-H1, CDR-H2 and / or CDR-H3 according to AbM number.

[0119] In some embodiments, the CAR contains an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment having a variable heavy chain (V H(a) CDR-H1, comprising an amino acid sequence selected from SEQ ID NO: 15, 18, 20, 22, 30, 33, 35, 37, 45, 48, 50, 52, 60, 63, 65, 67, 75, 78, 80, 82, 92, 102, 105, 107, and 109; (b) CDR-H2, comprising an amino acid sequence selected from SEQ ID NO: 16, 19, 21, 23, 31, 34, 36, 38, 46, 49, 51, 53, 61, 64, 66, 68, 76, 79, 81, 83, 90, 93, 94, 95, 103, 106, 108, and 110; and (c) CDR-H3, comprising an amino acid sequence selected from SEQ ID NO: The amino acid sequences of 17, 24, 32, 39, 47, 54, 62, 69, 77, 84, 91, 96, 104, and 111.

[0120] In some embodiments, the antibody or its antigen-binding fragment contains V H The area, the V HThe region contains CDR-H1, CDR-H2, and CDR-H3, each containing the following amino acid sequences: SEQ ID NO: 15, 16, and 17; SEQ ID NO: 18, 19, and 17; SEQ ID NO: 20, 21, and 17; SEQ ID NO: 22, 23, and 24; SEQ ID NO: 30, 31, and 32; SEQ ID NO: 33, 34, and 32; SEQ ID NO: 35, 36, and 32; SEQ ID NO: 37, 38, and 39; SEQ ID NO: 45, 46, and 47; SEQ ID NO: 48, 49, and 47; SEQ ID NO: 50, 51, and 47; SEQ ID NO: 52, 53, and 54; SEQ ID NO: 60, 61, and 62; SEQ ID NO: 63, 64, and 62; SEQ ID NO: 65, 66, and 62; SEQ ID NO: 67, 68, and 69; SEQ ID NO: 75, 76, and 77; SEQ ID NO: 78, 79, and 77; SEQ ID NO: 80, 81, and 77; SEQ ID NO: 82, 83, and 84; SEQ ID NO: 75, 90, and 91; SEQ ID NO: 92, 93, and 91; SEQ ID NO: 80, 94, and 91; SEQ ID NO: 82, 95, and 96; SEQ ID NO: 102, 103, and 104; SEQ ID NO: 105, 106, and 104; SEQ ID NO: 107, 108, and 104; or SEQ ID NO: 109, 110, and 111.

[0121] In some embodiments, the antibody or its antigen-binding fragment contains V H The area, the V HThe region contains the following amino acid sequences: respectively, SEQ ID NO: 15, 16 and 17; respectively, SEQ ID NO: 18, 19 and 17; respectively, SEQ ID NO: 20, 21 and 17; respectively, SEQ ID NO: 22, 23 and 24; respectively, SEQ ID NO: 30, 31 and 32; respectively, SEQ ID NO: 33, 34 and 32; respectively, SEQ ID NO: 35, 36 and 32; respectively, SEQ ID NO: 37, 38 and 39; respectively, SEQ ID NO: 45, 46 and 47; respectively, SEQ ID NO: 48, 49 and 47; respectively, SEQ ID NO: 50, 51 and 47; respectively, SEQ ID NO: 52, 53 and 54; respectively, SEQ ID NO: 60, 61 and 62; respectively, SEQ ID SEQ ID NOs: 63, 64, and 62; respectively, SEQ ID NOs: 65, 66, and 62; respectively, SEQ ID NOs: 67, 68, and 69; respectively, SEQ ID NOs: 75, 76, and 77; respectively, SEQ ID NOs: 78, 79, and 77; respectively, SEQ ID NOs: 80, 81, and 77; respectively, SEQ ID NOs: 82, 83, and 84; respectively, SEQ ID NOs: 75, 90, and 91; respectively, SEQ ID NOs: 92, 93, and 91; respectively, SEQ ID NOs: 80, 94, and 91; respectively, SEQ ID NOs: 82, 95, and 96; respectively, SEQ ID NOs: 102, 103, and 104; respectively, SEQ ID NOs: 105, 106, and 104; respectively, SEQ ID NOs: 107, 108, and 104; respectively, SEQ ID NOs: 108, 109, and 104; respectively, SEQ ID NOs: 100, 100, and 104; respectively, SEQ ID NOs: 100, 100, and 104; respectively, SEQ ID NOs: 100, 100, and 104; respectively, SEQ ID NOs: 100, 100, and 101 ... NO: 109, 110 and 111.

[0122] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1, CDR-H2, and CDR-H3 are respectively contained in the V shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13. H The amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 contained within the region's amino acid sequence.

[0123] In some embodiments of the antibody or its antigen-binding fragment provided herein, V HThe region comprises any of CDR-H1, CDR-H2, and CDR-H3 as described above and includes frame regions 1 (FR1), FR2, FR3, and / or FR4, wherein FR1, FR2, FR3, and / or FR4 are respectively associated with V shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13. H The amino acid sequences contained in the region contain FR1, FR2, FR3 and / or FR4 with 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.

[0124] In some embodiments, the antibody or its antigen-binding fragment comprises a V containing the amino acid sequence shown in any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13. H district.

[0125] In some implementations, V is included H The antibody or antibody fragment in the provided CAR (e.g., anti-GPRC5D CAR) further comprises a light chain or a sufficient antigen-binding portion thereof. For example, in some embodiments, the antibody or its antigen-binding fragment comprises V H District and V L District, or V H and V L Sufficient antigen-binding portion of the region. In such implementations, V H The region sequence can be any of the above V. H Sequence. In some such embodiments, the antibody is an antigen-binding fragment, such as Fab or scFv. In some such embodiments, the antibody is a full-length antibody that also contains a constant region.

[0126] In some embodiments, the antibody or antigen-binding fragment has the V described in any one of WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO 2019 / 154890, WO 2021 / 018925, WO 2018 / 147245 and WO 2023 / 134718. L district.

[0127] In some embodiments, the CAR contains an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment having a light chain variable (V) LThe light chain variable region has an amino acid sequence selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121 or 122, or a V sequence selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121 or 122. L The amino acids in the region have an amino acid sequence with 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; or contain amino acid sequences present in such a region. L The sequence contains CDR-L1, CDR-L2, and / or CDR-L3. In some embodiments, the CAR contains an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment having a variable light chain (V0). L The light chain variable region has an amino acid sequence selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, or a V sequence selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14. L The amino acids in the region have an amino acid sequence with 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; or contain amino acid sequences present in such a region. L The sequence contains CDR-L1, CDR-L2, and / or CDR-L3. In some embodiments, the CAR contains an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment having a variable light chain (V0). L The light chain variable region has an amino acid sequence selected from any one of SEQ ID NO: 116, 117, 118, 119, 120, 121 or 122, or a V sequence selected from any one of SEQ ID NO: 116, 117, 118, 119, 120, 121 or 122. L The amino acids in the region have an amino acid sequence with 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; or contain amino acid sequences present in such a region. LCDR-L1, CDR-L2 and / or CDR-L3 in the sequence.

[0128] In some implementations, the V of the antibody or its antigen-binding fragment L The region contains CDR-L1, CDR-L2, and / or CDR-L3 according to Kabat designations. In some embodiments, the V of the antibody or its antigen-binding fragment... L The region includes CDR-L1, CDR-L2, and / or CDR-L3 according to Chothia designations. In some embodiments, the V of the antibody or its antigen-binding fragment... L The region contains CDR-L1, CDR-L2 and / or CDR-L3 according to AbM numbering.

[0129] In some embodiments, the CAR contains an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment having a variable light chain (V0). L (a) CDR-L1, which contains amino acid sequences selected from SEQ ID NO: 25, 28, 40, 43, 55, 58, 70, 73, 85, 88, 97, 100, 112 and 114; (b) CDR-L2, which contains amino acid sequences selected from SEQ ID NO: 26, 29, 41, 44, 56, 59, 71, 74, 86, 89, 98 and 101; and (c) CDR-L3, which contains amino acid sequences selected from SEQ ID NO: 27, 42, 57, 72, 87, 99, 113 and 115.

[0130] In some embodiments, the antibody or its antigen-binding fragment contains V L The area, the V LThe region comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1, CDR-L2, and CDR-L3 respectively contain the following amino acid sequences: SEQ ID NO: 25, 26, and 27; SEQ ID NO: 28, 29, and 27; SEQ ID NO: 40, 41, and 42; SEQ ID NO: 43, 44, and 42; SEQ ID NO: 55, 56, and 57; SEQ ID NO: 58, 59, and 57; SEQ ID NO: 70, 71, and 72; SEQ ID NO: 73, 74, and 72; SEQ ID NO: 85, 86, and 87; SEQ ID NO: 88, 89, and 87; SEQ ID NO: 97, 98, and 99; SEQ ID NO: 100, 101, and 99; SEQ ID NO: 112, 26, and 113; SEQ ID NO: 114, 29 and 115; SEQ ID NO: 114, 29 and 123.

[0131] In some embodiments, the antibody or its antigen-binding fragment contains V L The area, the V L The region contains the following amino acid sequences: respectively, SEQ ID NO: 25, 26 and 27; respectively, SEQ ID NO: 28, 29 and 27; respectively, SEQ ID NO: 40, 41 and 42; respectively, SEQ ID NO: 43, 44 and 42; respectively, SEQ ID NO: 55, 56 and 57; respectively, SEQ ID NO: 58, 59 and 57; respectively, SEQ ID NO: 70, 71 and 72; respectively, SEQ ID NO: 73, 74 and 72; respectively, SEQ ID NO: 85, 86 and 87; respectively, SEQ ID NO: 88, 89 and 87; respectively, SEQ ID NO: 97, 98 and 99; respectively, SEQ ID NO: 100, 101 and 99; respectively, SEQ ID NO: 112, 26 and 129; respectively, SEQ ID NO: 114, 29 and 115; respectively, SEQ ID NO: 114, 29 and 123.

[0132] In some embodiments, the antibody or its antigen-binding fragment contains V, respectively, selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122. LThe region contains CDR-L1, CDR-L2, and CDR-L3 within its amino acid sequence. In some embodiments, the antibody or its antigen-binding fragment contains V, respectively, selected from any one of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. L The region contains CDR-L1, CDR-L2, and CDR-L3 within its amino acid sequence. In some embodiments, the antibody or its antigen-binding fragment contains V, respectively, selected from any one of SEQ ID NO: 116, 117, 118, 119, 120, 121, or 122. L The amino acid sequence in the region contains CDR-L1, CDR-L2, and CDR-L3.

[0133] The CARs provided in this article include the following CARs, wherein the antibody (such as anti-GPRC5D antibody or antibody fragment) in the provided CAR contains V H region amino acid sequence and V L The area, the V H The amino acid sequence of the region has 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 any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13; L The region contains an amino acid 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%, or about 98%, or at least about 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122.

[0134] In some implementations, the V of the antibody or its antigen-binding fragment H The region includes CDR-H1, CDR-H2, and CDR-H3, wherein CDR-H1, CDR-H2, and CDR-H3 are respectively contained in V selected from any one of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13. HThe amino acid sequence contains the amino acid sequences CDR-H1, CDR-H2, and CDR-H3; and includes CDR-L1, CDR-L2, and CDR-L3, wherein each of CDR-L1, CDR-L2, and CDR-L3 contains a V from any one of EQ ID NO: 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122. L The amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 contained within the region's amino acid sequence.

[0135] In some implementations, the V of the antibody or its antigen-binding fragment H The region contains the amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13, and the V of the antibody or antigen-binding fragment. L The region contains an amino acid sequence of 2, 4, 6, 8, 10, 12, 14, 116, 117, 118, 119, 120, 121, or 122. In some embodiments, the V of the antibody or its antigen-binding fragment... H and V L The regions respectively contain the following amino acid sequences: SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 4; SEQ ID NO: 5 and 6; SEQ ID NO: 7 and 8; SEQ ID NO: 9 and 10; SEQ ID NO: 11 and 12; or SEQ ID NO: 13 and 14, or the same as the above V. H and V L Any antibody or antigen-binding fragment thereof having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity). In other examples, the V of the antibody or antigen-binding fragment thereof provided herein. H and V L The region contains amino acid sequences selected from the following: SEQ ID NO: 1 and 116; SEQ ID NO: 3 and 117; SEQ ID NO: 5 and 118; SEQ ID NO: 7 and 119; SEQ ID NO: 9 and 120; SEQ ID NO: 11 and 121; or SEQ ID NO: 13 and 122, or the same as those described above. H and V L Any antibody or antigen-binding fragment thereof that has at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity).

[0136] For example, the V of the antibody or its antigen-binding fragment provided in this article H and V L The regions respectively contain amino acid sequences selected from the following: SEQ ID NO: 1 and 2; SEQ ID NO: 3 and 4; SEQ ID NO: 5 and 6; SEQ ID NO: 7 and 8; SEQ ID NO: 9 and 10; SEQ ID NO: 11 and 12; or SEQ ID NO: 13 and 14. In other examples, the V of the antibody or its antigen-binding fragment provided herein... H and V L The regions contain amino acid sequences selected from the following: SEQ ID NO: 1 and 116; SEQ ID NO: 3 and 117; SEQ ID NO: 5 and 118; SEQ ID NO: 7 and 119; SEQ ID NO: 9 and 120; SEQ ID NO: 11 and 121; or SEQ ID NO: 13 and 122.

[0137] In some embodiments, the antibody or its antigen-binding fragment in the provided CAR is a single-chain antibody fragment, such as a single-chain variable fragment (scFv), a bispecific antibody, or a single-domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment contains only V... H Single-domain antibodies in the region. In some embodiments, the antibody or antigen-binding fragment is a variable heavy chain (V... H ) region and light chain variable (V L The scFv of the ) region. In some embodiments, the single-chain antibody fragment (e.g., scFv) includes a region connecting two antibody domains or regions (such as the heavy chain variable (V) region). H ) region and light chain variable (V L One or more linkers in the region. Linkers are typically peptide linkers, such as flexible and / or soluble peptide linkers. Linkers include those rich in glycine and serine and / or, in some cases, threonine. In some embodiments, the linker also includes charged residues (such as lysine and / or glutamic acid) that can improve solubility. In some embodiments, the linker also includes one or more proline residues.

[0138] Therefore, the provided CAR contains an anti-GPRC5D antibody, which comprises single-chain antibody fragments, such as scFv, and biantibodies, particularly human single-chain antibody fragments, typically containing two antibody domains or regions (such as V...). H and V L One or more linkers in a region. Linkers are typically peptide linkers, such as flexible and / or soluble peptide linkers, such as a peptide linker rich in glycine and serine.

[0139] In some aspects, the linkers rich in glycine and serine (and / or threonine) 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% of glycine, serine, and / or threonine. In some embodiments, the linkers are substantially entirely composed of glycine, serine, and / or threonine. The length of the linker is typically between about 5 and about 50 amino acids, usually between 10 and about 30, for example 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and in some examples the length is between 10 and 25 amino acids. Exemplary linkers include linkers with different numbers of repeats of the sequence GGGGS (4GS; SEQ ID NO: 125) or GGGS (3GS; SEQ ID NO: 126) (such as repeats of this sequence between 2, 3, 4, and 5). Exemplary linkers include those having or consisting of the sequence shown in SEQ ID NO: 127 (GGGGSGGGGSGGGGS). Exemplary connectors also include those having or consisting of the sequence shown in SEQ ID NO: 128 (GSTSGSGKPGSGEGSTKG). Exemplary connectors also include those having or consisting of the sequence shown in SEQ ID NO: 129 (SRGGGGSGGGGSGGGGSLEMA). Exemplary connectors include those having or consisting of the sequence shown in SEQ ID NO: 124 (GSRGGGGSGGGGSGGGGSLEMA).

[0140] Therefore, in some embodiments, the provided embodiments include single-chain antibody fragments (e.g., scFv) containing one or more of the above-described adapters, such as glycine / serine-rich adapters (including adapters having GGGS (SEQ ID NO: 126) or GGGGS (SEQ ID NO: 125) repeats, such as the adapters shown in SEQ ID NO: 124, 127 or 129).

[0141] In some implementation schemes, V H The area can be located in V L The amino terminus of the region. In some embodiments, V H The area can be located in V L The carboxyl terminus of the region. In a particular embodiment, the fragment (e.g., scFv) may include V HThe area or part thereof, followed by the connector, followed by the V. L A region or a portion thereof. In other embodiments, a fragment (e.g., scFv) may include V. L The area or part thereof, followed by the connector, followed by the V. H The district or a part thereof.

[0142] In some respects, the scFv of the extracellular antigen-binding domain of the CAR provided herein comprises an amino acid sequence selected from any one of SEQ ID NO: 130-143 or 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 an amino acid sequence selected from any one of SEQ ID NO: 130-143.

[0143] In some respects, the scFv of the extracellular domain of the CAR provided herein is encoded by a nucleotide sequence selected from any one of SEQ ID NO: 144-157 or has a nucleotide 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 a nucleotide sequence selected from any one of SEQ ID NO: 144-157.

[0144] The provided anti-GPRC5D CAR includes the following CAR, wherein the antibody or antigen-binding fragment in the CAR contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 1 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% identity with SEQ ID NO: 1; the V LThe region contains the sequence shown in SEQ ID NO: 2 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% identity with SEQ ID NO: 2. In some embodiments, the provided CAR is a CAR in which the antibody or antigen binding fragment contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 1 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% identity with SEQ ID NO: 1; the V L The region contains the sequence shown in SEQ ID NO: 116 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% identity with SEQ ID NO: 116. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 15, 16, and 17, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO: 25, 26, and 27. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences of SEQ ID NO: 18, 19, and 17, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO: 25, 26, and 27. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the VH The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences of SEQ ID NO: 20, 21, and 17, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO: 25, 26, and 27. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 22, 23, and 24, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, which respectively contain the amino acid sequences of SEQ ID NO:28, 29, and 27. In some embodiments, V H The region contains the sequence shown in SEQ ID NO:1, and V L The region contains the sequence shown in SEQ ID NO: 2. In some embodiments, V H The region contains the sequence shown in SEQ IDNO: 1, and V LThe region contains the sequence shown in SEQ ID NO: 116. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as scFv. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 130 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% identity with SEQ ID NO: 130. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 144 or a nucleotide 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% identity with SEQ ID NO: 144. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 131 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% identity with SEQ ID NO: 131. In some embodiments, scFv is encoded by the nucleotide sequence shown in SEQ ID NO:145 or a nucleotide 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% identity with SEQ ID NO:145.

[0145] The provided anti-GPRC5D CAR includes the following CAR, wherein the antibody or antigen-binding fragment in the CAR contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 3 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% identity with SEQ ID NO: 3; the V LThe region contains the sequence shown in SEQ ID NO: 4 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% identity with SEQ ID NO: 4. In some embodiments, the provided CAR is a CAR in which the antibody or antigen binding fragment contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 3 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% identity with SEQ ID NO: 3; the V L The region contains the sequence shown in SEQ ID NO: 117 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% identity with SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 30, 31, and 32, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, comprising the amino acid sequences SEQ ID NO: 40, 41, and 42. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 33, 34, and 32, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 40, 41, and 42. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the VH The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 35, 36, and 32, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 40, 41, and 42. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 37, 38, and 39, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO:43, 44, and 42. In some embodiments, V H The region contains the sequence shown in SEQ ID NO:3, and V L The region contains the sequence shown in SEQ ID NO: 4. In some embodiments, V H The region contains the sequence shown in SEQ IDNO: 3, and V LThe region contains the sequence shown in SEQ ID NO: 117. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as scFv. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 132 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% identity with SEQ ID NO: 132. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 146 or a nucleotide 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% identity with SEQ ID NO: 146. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 133 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% identity with SEQ ID NO: 133. In some embodiments, scFv is encoded by the nucleotide sequence shown in SEQ ID NO:147 or a nucleotide 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% identity with SEQ ID NO:147.

[0146] The provided anti-GPRC5D CAR includes the following CAR, wherein the antibody or antigen-binding fragment in the CAR contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 5 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% identity with SEQ ID NO: 5; the V LThe region contains the sequence shown in SEQ ID NO: 6 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% identity with SEQ ID NO: 6. In some embodiments, the provided CAR is a CAR in which the antibody or antigen binding fragment contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 5 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% identity with SEQ ID NO: 5; the V L The region contains the sequence shown in SEQ ID NO: 118 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% identity with SEQ ID NO: 118. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 45, 46, and 47, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 55, 56, and 57. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 48, 49, and 47, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 55, 56, and 57. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the VH The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 50, 51, and 47, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 55, 56, and 57. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 52, 53, and 54, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO: 58, 59, and 57. In some embodiments, V H The region contains the sequence shown in SEQ ID NO:5, and V L The region contains the sequence shown in SEQ ID NO: 6. In some embodiments, V H The region contains the sequence shown in SEQ IDNO: 5, and V LThe region contains the sequence shown in SEQ ID NO: 118. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as scFv. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 134 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% identity with SEQ ID NO: 134. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 148 or a nucleotide 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% identity with SEQ ID NO: 148. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 135 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% identity with SEQ ID NO: 135. In some embodiments, scFv is encoded by the nucleotide sequence shown in SEQ ID NO:149 or a nucleotide 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% identity with SEQ ID NO:149.

[0147] The provided anti-GPRC5D CAR includes the following CAR, wherein the antibody or antigen-binding fragment in the CAR contains V H District and V L The area, the V H The region contains the 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% identity with SEQ ID NO: 7; the V LThe 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% identity with SEQ ID NO: 8. In some embodiments, the provided CAR is a CAR in which the antibody or antigen binding fragment contains V H District and V L The area, the V H The region contains the 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% identity with SEQ ID NO: 7; the V L The region contains the sequence shown in SEQ ID NO: 119 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% identity with SEQ ID NO: 119. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 60, 61, and 62, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO: 70, 71, and 72. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 63, 64, and 62, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 70, 71, and 72. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the VH The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 65, 66, and 62, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 70, 71, and 72. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 67, 68, and 69, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, which respectively contain the amino acid sequences SEQ ID NO:73, 74, and 72. In some embodiments, V H The region contains the sequence shown in SEQ ID NO:7, and V L The region contains the sequence shown in SEQ ID NO: 8. In some embodiments, V H The region contains the sequence shown in SEQ IDNO: 7, and V LThe region contains the sequence shown in SEQ ID NO: 119. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as scFv. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 136 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% identity with SEQ ID NO: 136. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 150 or a nucleotide 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% identity with SEQ ID NO: 150. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 137 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% identity with SEQ ID NO: 137. In some embodiments, scFv is encoded by the nucleotide sequence shown in SEQ ID NO:151 or a nucleotide 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% identity with SEQ ID NO:151.

[0148] The provided anti-GPRC5D CAR includes the following CAR, wherein the antibody or antigen-binding fragment in the CAR contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 9 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% identity with SEQ ID NO: 9; the V LThe region contains the sequence shown in SEQ ID NO: 10 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% identity with SEQ ID NO: 10. In some embodiments, the provided CAR is a CAR in which the antibody or antigen binding fragment contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 9 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% identity with SEQ ID NO: 9; the V L The region contains the sequence shown in SEQ ID NO: 120 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% identity with SEQ ID NO: 120. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 75, 76, and 77, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, comprising the amino acid sequences SEQ ID NO: 85, 86, and 87. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 78, 79, and 77, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 85, 86, and 87. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the VH The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 80, 81, and 77, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 85, 86, and 87. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 82, 83, and 84, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 88, 89, and 87. In some embodiments, V H The region contains the sequence shown in SEQ ID NO:9, and V L The region contains the sequence shown in SEQ ID NO: 10. In some embodiments, V H The region contains the sequence shown in SEQ ID NO: 9, and V LThe region contains the sequence shown in SEQ ID NO: 120. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as scFv. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 138 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% identity with SEQ ID NO: 138. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 152 or a nucleotide 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% identity with SEQ ID NO: 152. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 139 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% identity with SEQ ID NO: 139. In some embodiments, scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 153 or a nucleotide 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% identity with SEQ ID NO: 153.

[0149] The provided anti-GPRC5D CAR includes the following CAR, wherein the antibody or antigen-binding fragment in the CAR contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 11 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% identity with SEQ ID NO: 11; the V LThe region contains the sequence shown in SEQ ID NO: 12 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% identity with SEQ ID NO: 12. In some embodiments, the provided CAR is a CAR in which the antibody or antigen binding fragment contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 11 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% identity with SEQ ID NO: 11; the V L The region contains the sequence shown in SEQ ID NO: 121 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% identity with SEQ ID NO: 121. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 75, 90, and 91, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, comprising the amino acid sequences SEQ ID NO: 97, 98, and 99. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 92, 93, and 91, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 97, 98, and 99. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V LThe area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively, containing the amino acid sequences SEQ ID NO: 80, 94, and 91, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 97, 98, and 99. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 82, 95, and 96, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 100, 101, and 99. In some embodiments, V H The region contains the sequence shown in SEQ ID NO: 11, and V L The region contains the sequence shown in SEQ ID NO: 12. In some embodiments, V H The region contains the sequence shown in SEQ ID NO: 11, and V LThe region contains the sequence shown in SEQ ID NO: 121. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as scFv. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 140 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% identity with SEQ ID NO: 140. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 154 or a nucleotide sequence having at least or at least 90%, at least or at least 91%, at least or at least 92%, at least or at least 93%, at least or at least 94%, at least or at least 95%, at least or at least 96%, at least or at least 97%, at least or at least 98%, or at least or at least 99% identity with SEQ ID NO: 154. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 141 or an amino acid sequence having at least or at least 90%, at least or at least 91%, at least or at least 92%, at least or at least 93%, at least or at least 94%, at least or at least 95%, at least or at least 96%, at least or at least 97%, at least or at least 98%, or at least or at least 99% identity with SEQ ID NO: 141. In some embodiments, scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 155 or a nucleotide 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% identity with SEQ ID NO: 155.

[0150] The provided anti-GPRC5D CAR includes the following CAR, wherein the antibody or antigen-binding fragment in the CAR contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 13 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% identity with SEQ ID NO: 13; the V LThe region contains the sequence shown in SEQ ID NO: 14 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% identity with SEQ ID NO: 14. In some embodiments, the provided CAR is a CAR in which the antibody or antigen binding fragment contains V H District and V L The area, the V H The region contains the sequence shown in SEQ ID NO: 13 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% identity with SEQ ID NO: 13; the V L The region contains the sequence shown in SEQ ID NO: 122 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% identity with SEQ ID NO: 122. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 102, 103, and 104, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO: 112, 26, and 113. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 105, 106, and 104, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 112, 26, and 113. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and VL The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 107, 108, and 104, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 112, 26, and 113. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 109, 110, and 111, wherein the V L The region comprises CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences of SEQ ID NO: 114, 29, and 115. In some embodiments, the antibody or antigen-binding fragment of the provided CAR contains V H District and V L The area, the V H The region comprises CDRH1, CDRH2, and CDRH3, respectively containing the amino acid sequences SEQ ID NO: 109, 110, and 111, wherein the V L The region contains CDRL1, CDRL2, and CDRL3, respectively, containing the amino acid sequences SEQ ID NO: 114, 29, and 123. In some embodiments, V H The region contains the sequence shown in SEQ ID NO: 13, and V L The region contains the sequence shown in SEQ ID NO: 14. In some embodiments, V H The region contains the sequence shown in SEQ ID NO: 13, and V LThe region contains the sequence shown in SEQ ID NO: 122. In some embodiments, the antibody or antigen-binding fragment is a single-chain antibody fragment, such as scFv. In some embodiments, the scFv contains the amino acid sequence shown in SEQ ID NO: 142 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% identity with SEQ ID NO: 142. In some embodiments, the scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 156 or a nucleotide sequence having at least or at least 90%, at least or at least 91%, at least or at least 92%, at least or at least 93%, at least or at least 94%, at least or at least 95%, at least or at least 96%, at least or at least 97%, at least or at least 98%, or at least or at least 99% identity with SEQ ID NO: 156. In some embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO: 143 or an amino acid sequence having at least or at least 90%, at least or at least 91%, at least or at least 92%, at least or at least 93%, at least or at least 94%, at least or at least 95%, at least or at least 96%, at least or at least 97%, at least or at least 98%, or at least or at least 99% identity with SEQ ID NO: 143. In some embodiments, scFv is encoded by the nucleotide sequence shown in SEQ ID NO: 157 or a nucleotide 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% identity with SEQ ID NO: 157. b. Spacer

[0151] In some embodiments, the recombinant receptor (such as a CAR) comprising an antibody (e.g., an antigen-binding fragment) provided herein further comprises a spacer, which may be or include at least a portion of an immunoglobulin constant region or a variant or modified form thereof. In some embodiments, the portion of the immunoglobulin constant region includes a hinge region, such as an IgG4 hinge region and / or C H 1. C H 2 or C H3 and / or the Fc region. In some embodiments, the constant region or a portion thereof is human IgG (such as IgG4 or IgG1). In some aspects, a portion of the constant region serves as a spacer region between an antigen recognition component (such as an antigen-binding domain (e.g., scFv)) and a transmembrane domain. In some embodiments, the length of the spacer is adjusted to optimize the biophysical synaptic distance between CAR-expressing cells (such as CAR-expressing cells) and the CAR's target (such as GPRC5D-expressing tumor cells). In some embodiments, the CAR is expressed by T cells, and the length of the spacer is adjusted to a length compatible with T cell activation or is adjusted to optimize CAR T cell performance.

[0152] In some embodiments, the spacer may have a length that provides increased cellular responsiveness upon antigen binding compared to the absence of a spacer or compared to alternative spacers of different lengths (e.g., shorter lengths). In some examples, the spacer length is one or about 12 amino acids, or no more than 12 amino acids. In some embodiments, the spacer has a length of at least 100 amino acids, such as 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 spacer region has a length of 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.

[0153] In some embodiments, the spacer has a length of 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 spacer has a length of at least or at least about or has or has about 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228 or 229 amino acids or a length between any of the foregoing values.

[0154] Exemplary spacers include individual IgG hinges, with C H 2 and C H One or more IgG hinges connected in the 3-domain structure or with C H 3. IgG hinge with connected structural domains. In some implementations, the IgG hinge, C... H 2 and / or C H 3 can be wholly or partially derived from IgG4 or IgG2, such as wholly or partially derived from human IgG4 or human IgG2. In some embodiments, the spacer may be a hinge containing IgG4, IgG2, and / or IgG2 and IgG4, C H 2 and / or C H One or more chimeric polypeptides in sequence 3. In some embodiments, the hinge region comprises all or a portion 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 Region 2 contains IgG4 C H Region 2 and / or IgG2 C H All or part of region 2, in which IgG4 C H Zone 2 is a random location for human IgG4 C H Region 2, and IgG2 C H Zone 2 is a random location for human IgG2 CH Zone 2; and / or C H Region 3 contains IgG4 C H 3 regions and / or IgG2 C H All or part of region 3, including IgG4 C H The three regions are randomly selected for human IgG4 C. H Region 3, and IgG2 C H The three regions are randomly selected for human IgG2 C. H Zone 3. In some implementations, hinge C H 2 and C H 3 contains the hinge region C from IgG4 H 2 and C H All or part of each of 3. In some embodiments, the hinge region is interlocking and includes hinge regions from human IgG4 and human IgG2; the C H Region 2 is chimeric and contains C from human IgG4 and human IgG2. H Zone 2; and / or the aforementioned C H Region 3 is chimeric and contains C from human IgG4 and human IgG2. H Region 3. In some embodiments, the spacer comprises an IgG4 / 2 chimeric hinge or a modified IgG4 hinge containing at least one amino acid substitution compared to the human IgG4 hinge region; human IgG2 / 4 chimeric C H Zone 2; and human IgG4 C H Zone 3.

[0155] 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 examples, the amino acid modification is the substitution of serine (S) with proline (P) 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, as shown in the C of the full-length IgG4 Fc sequence of SEQ ID NO: 158. H The N177Q mutation at position 177 in region 2, or the C mutation in the full-length IgG2 Fc sequence shown in SEQ ID NO: 159. H N176Q at position 176 in region 2. In some embodiments, the spacer is or contains an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; IgG2 / 4 chimeric C H Region 2; and IgG4 C HRegion 3. In some embodiments, the spacer is approximately 228 amino acids long. In some embodiments, the spacer is as shown in SEQ ID NO: 162. In some embodiments, the spacer contains an amino acid sequence. ESKYGPPCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 162)

[0156] In some embodiments, the spacer is encoded by a polynucleotide optimized for codon expression and / or elimination of splicing sites (such as cryptic splicing sites). In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO: 164. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO: 163. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO: 165. In some embodiments, the coding sequence of the spacer comprises the nucleic acid sequence shown in SEQ ID NO: 166.

[0157] Other exemplary spacers include, but are not limited to, those described in the following literature: 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. WO 2014031687. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce post-expression RNA heterogeneity. In some embodiments, the nucleotide sequence of the spacer is optimized to reduce cryptic splicing sites or reduce the likelihood of splicing events occurring at splicing sites.

[0158] In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 160 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 167. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 161. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 168.

[0159] In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 162, which is encoded by the polynucleotide sequence shown in SEQ ID NO: 163, 164, 165 or 166 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 with SEQ ID NO: 163, 164, 165 or 166.

[0160] In some embodiments, the spacer has an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with SEQ ID NO: 162, encoded by a polynucleotide that has optionally been optimized for codon usage and / or to reduce RNA heterogeneity. Methods for reducing RNA heterogeneity include, for example, removing cryptic splicing donor and / or receptor sites, as described in section IA2.b below. Observations have shown that, when present in a CAR, cryptic splicing donor and / or receptor sites are present in the spacer region of certain immunoglobulin spacers. In some embodiments, the spacer in the provided CAR is encoded by a polynucleotide in which one or more cryptic splicing donor and / or receptor sites are eliminated and / or modified to reduce the heterogeneity of RNA (e.g., mRNA) transcribed from the construct after expression in the cell. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 164 (also shown in SEQ ID NO: 171). In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 165. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 166. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 172.

[0161] In some embodiments, the spacer contains a hinge domain from CD28. In some embodiments, the spacer has the amino acid sequence shown in SEQ ID NO: 170 and is encoded by the polynucleotide sequence shown in SEQ ID NO: 169. c. Transmembrane domains and intracellular signal transduction components

[0162] Antigen recognition components are typically linked to one or more intracellular signaling components (e.g., signaling components that mimic activation via an antigen receptor complex (e.g., a TCR complex in the case of a CAR) and / or signal transduction via another cell surface receptor). Therefore, in some embodiments, a GPRC5D binding molecule (e.g., an antibody or its antigen-binding fragment) is linked to one or more transmembrane domains (as described herein) and an intracellular signaling domain containing one or more intracellular components (as described herein). In some embodiments, the transmembrane domain is fused to an extracellular domain. In one embodiment, a transmembrane domain that naturally associates with one of the domains in a receptor (e.g., a CAR) is used. In some cases, transmembrane domains are selected or modified by amino acid substitution to prevent such domains from binding to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex.

[0163] In some embodiments, the transmembrane domain is derived from a natural or synthetic source. When the source is natural, in some aspects, the domain may be derived from any membrane-binding protein or transmembrane protein. Transmembrane domains include those derived from (i.e., transmembrane domains containing at least one or more of the following): the α, β, or ζ chain 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, the transmembrane domain may be a CD28 transmembrane domain containing the amino acid sequence shown in SEQ ID NO: 173, which is encoded by the nucleic acid sequence shown in SEQ ID NO: 174 or SEQ ID NO: 175. Alternatively, in some embodiments, the transmembrane domain is synthetic. In some aspects, synthetic transmembrane domains primarily contain hydrophobic residues, such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine will be found at each end of the synthetic transmembrane domain. In some implementations, the connection is achieved through a linker, a spacer, and / or one or more transmembrane domains.

[0164] Intracellular signal transduction domains include those that mimic or approximate the following: signaling via a natural antigen receptor, signaling via a combination of such receptor and a co-stimulatory receptor, and / or signaling via a co-stimulatory receptor alone. In some embodiments, short oligopeptide or polypeptide linkers (e.g., linkers of 2 to 10 amino acids in length, such as linkers containing glycine and serine, e.g., glycine-serine dinucleotides) are present and form a connection between the transmembrane domain of the CAR and the intracellular signal transduction domain.

[0165] Receptors (e.g., CARs) typically include an intracellular signaling domain containing at least one or more intracellular signaling components. In some embodiments, the receptor comprises an intracellular component or signaling domain of the TCR complex, such as the TCR CD3 chain mediating T cell activation and cytotoxicity, for example, the CD3ζ (CD3-ζ) chain. Therefore, in some aspects, the GPRC5D 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 (e.g., CAR) also comprises a portion of one or more other molecules, such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR comprises a CD3-ζ (CD3-ζ) or a chimeric molecule between Fc receptor γ and CD8, CD4, CD25, or CD16.

[0166] In some embodiments, upon CAR conjugation, the cytoplasmic domains or intracellular signaling domains of the CAR stimulate and / or activate at least one of the normal effector functions or responses of immune cells (e.g., T cells engineered to express the CAR). For example, in some contexts, the CAR induces T cell functions such as cytolytic activity or T helper 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 (e.g., if it transduces effector function signals) is used instead of the complete immune stimulation chain. In some embodiments, one or more intracellular signaling domains comprise a cytoplasmic sequence of a T cell receptor (TCR) and, in some respects, also include co-receptors (which in the natural context synergize with such receptors to initiate signal transduction upon antigen receptor conjugation) and / or any derivatives or variants of such molecules, and / or any synthetic sequence having the same functional capabilities.

[0167] In the context of a natural TCR, full activation typically requires not only signal transduction via the TCR but also co-stimulatory signals. Therefore, in some embodiments, components for generating secondary or co-stimulatory signals are also included in the CAR to promote full activation. In other embodiments, the CAR does not contain components for generating co-stimulatory signals. In some aspects, an additional CAR is expressed in the same cell and provides components for generating secondary or co-stimulatory signals.

[0168] In some respects, T cell activation is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in a non-antigen-dependent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences). In some respects, CARs contain one or both of these cytoplasmic signaling sequences.

[0169] In some aspects, the CAR contains a primary cytoplasmic signaling sequence that regulates primary stimulation and / or activation of the TCR complex. The primary cytoplasmic signaling sequence acting in a stimulatory manner may contain a signaling motif called an immune receptor tyrosine activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include those derived from TCR or CD3ζ, FcRγ, CD3γ, CD3δ, and CD3ε. In some embodiments, the intracellular signaling region in the CAR contains a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ. In some embodiments, CD3ζ comprises the amino acid sequence shown in SEQ ID NO:176, encoded by the nucleic acid sequence shown in SEQ ID NO:177 or SEQ ID NO:178.

[0170] In some embodiments, the CAR comprises a signal transduction domain (e.g., an intracellular or cytoplasmic signal transduction domain) and / or a transmembrane portion of a co-stimulatory molecule (such as 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 comprise the amino acid sequence shown in SEQ ID NO: 179, encoded by the nucleotide sequence shown in SEQ ID NO: 180 or SEQ ID NO: 181. In some aspects, the same CAR comprises both a stimulatory or activating component (e.g., a cytoplasmic signal transduction sequence) and a co-stimulatory component.

[0171] In some embodiments, the stimulatory or activating component is contained in one CAR, while the co-stimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR contains both an activating or stimulatory CAR and a co-stimulatory CAR expressed on the same cell (see WO 2014 / 055668). In some respects, the CAR targeting GPRC5D is a stimulatory or activating CAR; in others, it is a co-stimulatory CAR. In some embodiments, the cell also contains an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013)), such as a CAR that recognizes antigens other than GPRC5D, thereby reducing or inhibiting the stimulatory or activating signal delivered by the CAR targeting GPRC5D through the binding of the inhibitory CAR to its ligand, for example, to reduce off-target effects.

[0172] In some embodiments, the intracellular signaling region includes a CD28 transmembrane and signaling domain connected to the intracellular domain of CD3 (e.g., CD3-ζ). In some embodiments, the intracellular signaling domain includes a chimeric CD28 and 4-1BB (CD137; TNFRSF9) co-stimulatory domain connected to the intracellular domain of CD3ζ.

[0173] In some implementations, the CAR encompasses one or more (e.g., two or more) co-stimulatory domains and stimulatory or activating domains (e.g., primary activating domains) in the cytoplasm. Exemplary CARs include intracellular components of CD3ζ, CD28, and 4-1BB.

[0174] In some embodiments, the provided anti-GPRC5D CAR includes an extracellular antigen-binding domain containing any anti-GPRC5D antibody or antigen-binding fragment described herein (such as in Section I.A1); containing an IgG4 / 2 chimeric hinge or a modified IgG4 hinge, or an IgG2 / 4 chimeric C H Region 2 and IgG4 C HThe spacer in region 3, such as a spacer of about 228 amino acids in length, or the spacer shown in SEQ ID NO: 162, encoded by a nucleotide sequence shown in any one of SEQ ID NO: 163, 164, 165, or 166; a transmembrane domain, such as a transmembrane domain from human CD28; and an intracellular signaling region comprising a cytoplasmic signaling domain of the CD3-ζ (CD3ζ) chain and an intracellular signaling domain of the T cell costimulatory molecule. A polynucleotide encoding such a chimeric antigen receptor is also provided. In some embodiments, the transmembrane domain is or comprises the sequence shown in SEQ ID NO: 173. In some embodiments, the intracellular signaling domain of the T cell costimulatory molecule is an intracellular signaling domain of human CD28, human 4-1BB, or human ICOS, or a signaling portion thereof. In a particular embodiment, the intracellular signaling domain is the intracellular signaling domain of human 4-1BB. In some embodiments, the intracellular signal transduction domain is or includes the sequence shown in SEQ ID NO: 179. In some embodiments, the cytoplasmic signal transduction domain is the human CD3-ζ cytoplasmic signal transduction domain, as shown in SEQ ID NO: 176. In some embodiments, the intracellular signal transduction region includes the sequences shown in SEQ ID NO: 176 and SEQ ID NO: 179.

[0175] In some embodiments, the provided anti-GPRC5D CAR embodiment has the amino acid sequence shown in SEQ ID NO: 183, 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: 182. In some embodiments, the provided anti-GPRC5D CAR embodiment is encoded by the nucleotide sequence shown in SEQ ID NO: 182, or a nucleotide 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 sequence shown in SEQ ID NO: 182.

[0176] In some embodiments, the provided anti-GPRC5D CAR embodiment has the amino acid sequence shown in SEQ ID NO: 208, 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: 209. In some embodiments, the provided anti-GPRC5D CAR embodiment is encoded by the nucleotide sequence shown in SEQ ID NO: 209, or a nucleotide 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 sequence shown in any of SEQ ID NO: 209. 2. Nucleic acids, vectors, and methods for genetic engineering

[0177] In some embodiments, cells (e.g., T cells) are genetically engineered to express the recombinant receptor. In some embodiments, engineering is performed by introducing a polynucleotide encoding the recombinant receptor. Polynucleotides encoding the recombinant receptor, as well as vectors or constructs containing such nucleic acids and / or polynucleotides, are also provided.

[0178] In some cases, the nucleic acid sequence encoding a recombinant receptor contains a signal sequence encoding a signal peptide. In some respects, the signal sequence can encode a signal peptide derived from a natural polypeptide. In other respects, the signal sequence can encode a heterologous or non-natural signal peptide. In some cases, the nucleic acid sequence encoding a recombinant receptor (e.g., a chimeric antigen receptor (CAR)) contains a signal sequence encoding a signal peptide.

[0179] In some embodiments, the polynucleotide encoding the recombinant receptor contains at least one promoter operatively linked to control the expression of the recombinant receptor. In some examples, the polynucleotide contains two, three, or more promoters operatively linked to control the expression of the recombinant receptor.

[0180] In some cases where nucleic acid molecules encode two or more different polypeptide chains (e.g., recombinant receptors and markers), each polypeptide chain may be encoded by a separate nucleic acid molecule. For example, two separate nucleic acids are provided, and each can be separately transferred into or introduced into a cell for expression. In some embodiments, the nucleic acid encoding the recombinant receptor and the nucleic acid encoding the marker are operatively linked to the same promoter and optionally separated by an internal ribosome entry site (IRES) or a nucleic acid encoding a self-cleaving peptide or a ribosome-jumping peptide (optionally T2A, P2A, E2A, or F2A). In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are operatively linked to two different promoters. In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are present or inserted at different locations within the cellular genome. In some embodiments, the polynucleotide encoding the recombinant receptor is introduced into a composition containing cultured cells, such as by retroviral transduction, transfection, or transformation.

[0181] In some embodiments, such as those where the polynucleotide contains first and second nucleic acid sequences, the coding sequence encoding each of the different polypeptide chains can be operatively linked to the same or different promoters. In some embodiments, the nucleic acid molecule can contain a promoter that drives the expression of two or more different polypeptide chains. In some embodiments, such nucleic acid molecules can be polycistronic (bicistronic or tricistronic, see, for example, U.S. Patent No. 6,060,273). In some embodiments, the transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows co-expression of gene products (e.g., encoding a marker and encoding a recombinant receptor) via information from a single promoter. Alternatively, in some cases, a single promoter can guide the expression of RNA containing two or three genes (e.g., encoding a marker and encoding a recombinant receptor) within a single open reading frame (ORF), the genes being separated from each other by a sequence encoding a self-cleaving peptide (e.g., a 2A sequence) or a protease recognition site (e.g., furin). Therefore, the ORF encodes a single polypeptide, which is processed into a separate protein during translation (in the case of 2A) or post-translational. In some cases, the peptide (such as T2A) can cause the ribosome to skip the synthesis of the peptide bond at the C-terminus of the 2A element (ribosome skipping), resulting in the end of the 2A sequence being separated from the next downstream peptide (see, for example, de Felipe, GeneticVaccines and Ther. 2:13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from viruses such as foot-and-mouth disease virus (F2A), equine rhinitis virus (E2A), *Tetrasomy taeniae* β-virus (T2A), and porcine swine vesicle virus-1 (P2A), as described in U.S. Patent Publication No. 20070116690.

[0182] Any recombinant receptor described herein may be encoded by a polynucleotide containing one or more nucleic acid sequences encoding the recombinant receptor, said nucleic acid sequences in any combination or arrangement. For example, one, two, three or more polynucleotides may encode one, two, three or more different polypeptides, such as recombinant receptors. In some embodiments, a vector or construct contains a nucleic acid sequence encoding a marker, and a separate vector or construct contains a nucleic acid sequence encoding a recombinant receptor (e.g., CAR). In some embodiments, the nucleic acid encoding the marker and the nucleic acid encoding the recombinant receptor are operatively linked to two different promoters. In some embodiments, the nucleic acid encoding the recombinant receptor is located downstream of the nucleic acid encoding the marker.

[0183] In some embodiments, the vector backbone contains a nucleic acid sequence encoding one or more markers. In some embodiments, the one or more markers are transduction markers, alternative markers, and / or selection markers.

[0184] In some embodiments, the marker is a transduction marker or a substitute marker. Transduction markers or substitute markers can be used to detect cells in which a polynucleotide (e.g., a polynucleotide encoding a recombinant receptor) has been introduced. In some embodiments, the transduction marker can indicate or confirm modifications to the cell. In some embodiments, the substitute marker is a protein prepared to be co-expressed with the recombinant receptor (e.g., CAR) on the cell surface. In certain embodiments, such a substitute marker is a surface protein modified to have minimal or no activity. In some embodiments, the substitute marker is encoded by the same polynucleotide encoding the recombinant receptor. In some embodiments, the nucleic acid sequence encoding the recombinant receptor is operatively linked to the nucleic acid sequence encoding the marker, optionally separated by an internal ribosome entry site (IRES) or by nucleic acids encoding a self-cleaving peptide or a peptide that causes ribosome jumping (e.g., a 2A sequence, such as T2A, P2A, E2A, or F2A). In some cases, engineered cells can be combined with non-inherent marker genes to allow for cell detection or selection, and in some cases, also to promote cell suicide.

[0185] Exemplary alternative markers may include truncated forms of cell surface peptides, such as those that are nonfunctional and do not transduce or cannot transduce signals, or signals that are typically transduced by the full-length form of the cell surface peptide, and / or are not internalized or cannot be internalized. Exemplary truncated cell surface peptides include truncated forms of growth factors or other receptors, such as truncated human epidermal growth factor receptor 2 (tHER2), truncated epidermal growth factor receptor (tEGFR, the exemplary tEGFR sequence listed in SEQ ID NO: 254 or 255), or prostate-specific membrane antigen (PSMA), or modified forms thereof. tEGFR may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibodies or binding molecules, which can be used to identify or select cells engineered with tEGFR constructs and encoded exogenous proteins, and / or to eliminate or isolate cells expressing the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34(4): 430-434. In some aspects, the markers (e.g., alternative markers) include all or part (e.g., truncated forms) of CD34, NGFR, CD19, or truncated CD19 (e.g., truncated nonhuman CD19) or epidermal growth factor receptor (e.g., tEGFR).

[0186] In some embodiments, the marker is or includes fluorescent proteins such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP) (e.g., hyperfolded GFP (sfGFP)), red fluorescent protein (RFP) (e.g., tdTomato, mCherry, mStrawberry, AsRed2, DsRed, or DsRed2), cyan fluorescent protein (CFP), blue-green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of said fluorescent proteins. In some embodiments, the marker is or includes an enzyme (e.g., luciferase), the lacZ gene from *E. coli*, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). Exemplary luminescent reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), or variants thereof.

[0187] In some embodiments, the marker is a selection marker. In some embodiments, the selection marker is or includes a polypeptide that confers resistance to a foreign agent or drug. In some embodiments, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that confers antibiotic resistance to mammalian cells. In some embodiments, the selection marker is or includes a puromycin resistance gene, a hygromycin resistance gene, an isoprothiolane resistance gene, a neomycin resistance gene, a genimycin resistance gene, or a geomycin resistance gene, or a modified form thereof.

[0188] In some implementations, the molecule is a non-self molecule, such as a non-self protein, which is a molecule that is not recognized as "self" by the immune system of the host to which the cell is to be adopted.

[0189] In some embodiments, the marker does not provide any therapeutic function and / or produces any effect other than serving as a marker for genetic engineering (e.g., for selecting successfully engineered cells). In other embodiments, the marker may be a therapeutic molecule or a molecule that otherwise performs some of the desired effects, such as a ligand for cells encountered in vivo, or a co-stimulatory or immune checkpoint molecule used to enhance and / or weaken cellular responses upon adoptive transfer and encounter with the ligand.

[0190] In some embodiments, the nucleic acid encoding the marker is operatively linked to a polynucleotide encoding an adapter sequence (such as a cleavable adapter sequence, e.g., T2A). For example, the marker and optional adapter sequence can be any of those disclosed in PCT Publication No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) optionally linked to an adapter sequence, such as a T2A cleavable adapter sequence. Exemplary polypeptides of truncated EGFR (e.g., tEGFR) comprise the amino acid sequence shown in SEQ ID NO: 254 or 255, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity with SEQ ID NO: 254 or 255.

[0191] In some implementations, recombinant nucleic acids are transferred to cells using recombinant infectious viral particles (e.g., vectors derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV)). In some implementations, recombinant nucleic acids are transferred to T cells using recombinant lentiviral vectors or retroviral vectors (e.g., 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(11): 550-557).

[0192] In some implementations, the vector is adeno-associated virus (AAV).

[0193] In some embodiments, the retroviral vector has a long terminal repeat (LTR) sequence, such as those derived from Moloni murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), and spleen lesion-forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses are typically amphiphilic, 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. Numerous exemplary retroviral systems have been described (e.g., U.S. Patent Nos. 5,219,740, 6,207,453, 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) HumanGene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109).

[0194] In some implementations, cells (e.g., T cells) can be transfected during or after amplification, for example with a T-cell receptor (TCR) or a chimeric antigen receptor (CAR). For example, this transfection of the gene for introducing the desired receptor can be performed using any suitable retroviral vector. The genetically modified cell population can then be evacuated from the initial stimulus (e.g., an anti-CD3 / anti-CD28 stimulus) and subsequently stimulated with a second type of stimulus, for example, via a de novo introduced receptor. This second type of stimulus can include antigenic stimuli in the form of peptide / MHC molecules, homologous (crosslinked) ligands of the genetically introduced receptor (e.g., natural ligands of the CAR), or any ligand (e.g., antibodies) that bind directly within the framework of the new receptor (e.g., by recognizing a constant region within the receptor). See, for example, Cheadle et al., “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).

[0195] In some cases, vectors that do not require cell activation (e.g., T cells) can be used. In some such cases, cells can be selected and / or transduced prior to activation. Therefore, cells can be engineered before or after cell culture, and in some cases, at the same time or at least for a portion of the culture period.

[0196] Other nucleic acids (e.g., genes for introduction) include those for improving therapeutic efficacy, such as by promoting the viability and / or function of metastatic cells; genes that provide genetic markers for selecting and / or evaluating cells (e.g., for assessing in vivo survival or localization); genes for improving safety, such as by making cells sensitive to negative selection in vivo, as described in Lupton SD et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human GeneTherapy 3:319-338 (1992); see also the disclosures of Lupton et al., PCT / US91 / 08442 and PCT / US94 / 05601, which describe the use of bifunctional selective fusion genes obtained by fusing a dominant positive selectivity marker with a negative selectivity marker. See, for example, Riddell et al., U.S. Patent No. 6,040,177, columns 14-17. a. Polynucleotides

[0197] Also provided are polynucleotides encoding the chimeric antigen receptor and / or portions thereof (e.g., chains). The provided polynucleotides include those encoding the anti-GPRC5D chimeric antigen receptor (e.g., antigen-binding fragment) described herein. Polynucleotides may include those comprising naturally occurring and / or non-naturally occurring nucleotides and bases, such as those having backbone modifications. The terms “nucleic acid molecule,” “nucleic acid,” and “polynucleotide” are used interchangeably and refer to polymers of nucleotides. Such polymers of nucleotides may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A “nucleic acid sequence” refers to a linear sequence of nucleotides constituting a nucleic acid molecule or polynucleotide.

[0198] In some cases, the polynucleotide encoding the GPRC5D binding receptor contains a signal sequence encoding a signal peptide, which in some cases is encoded upstream of the nucleic acid sequence encoding the GPRC5D binding receptor or at the 5' end of the nucleic acid sequence encoding the antigen-binding domain. In some cases, the polynucleotide containing the nucleic acid sequence encoding the GPRC5D binding receptor (e.g., a chimeric antigen receptor (CAR)) contains a signal sequence encoding a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a natural polypeptide. In other aspects, the signal sequence may encode a heterologous or non-natural signal peptide. In some aspects, non-limiting exemplary signal peptides include signal peptides of the IgGκ chain, as shown in SEQ ID NO: 185 or encoded by the nucleotide sequences shown in SEQ ID NO: 184 or 186-189. In some aspects, non-limiting exemplary signal peptides include signal peptides of the GMCSFRα chain, as shown in SEQ ID NO: 191 and encoded by the nucleotide sequence shown in SEQ ID NO: 190. In some aspects, non-limiting exemplary signal peptides include signal peptides of the CD8α signal peptide shown in SEQ ID NO: 192. In some aspects, non-limiting exemplary signal peptides include the signal peptide of the CD33 signal peptide shown in SEQ ID NO: 193. In some cases, the polynucleotide encoding the GPRC5D 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 GPRC5D binding receptor may be operatively linked to any additional component.

[0199] This document also provides codon-differentiated polynucleotide constructs encoding CARs. In some embodiments, the polynucleotide construct encodes a CAR capable of binding to GPRC5D, such as any CAR described herein. In some embodiments, cells express an anti-GPRC5D CAR as a therapeutic agent against multiple myeloma plasma cells. In some embodiments, the polynucleotide construct is codon-differentiated to improve the expression of the CAR encoded by the polynucleotide.

[0200] In some embodiments, the CARs provided herein include those encoded by polynucleotides that are optimized or contain certain features designed for optimization, such as optimization for codon usage, to reduce RNA heterogeneity and / or modify (e.g., augment or confer) the encoded receptor for more consistent expression (e.g., surface expression) across batches of cell products. In some embodiments, the polynucleotide encoding a GPRC5D-binding cell surface protein is modified, for example, to remove cryptic or hidden splicing sites, thereby reducing RNA heterogeneity compared to a reference polynucleotide. In some embodiments, the polynucleotide encoding a GPRC5D-binding cell surface protein is codon-optimized, such as for expression in mammalian (e.g., human) cells (e.g., human T cells). In some aspects, when expressed in cells, the modified polynucleotide results in improved, for example, increased or more uniform or consistent levels of expression (e.g., surface expression). Such polynucleotides can be used in constructs to generate engineered cells that express the GPRC5D-binding cell surface protein. Therefore, cells expressing the recombinant receptor encoded by the polynucleotides provided herein are also provided, and their use in adoptive cell therapy, such as the treatment of diseases and disorders associated with GPRC5D expression (e.g., multiple myeloma).

[0201] Also provided are cells (such as T cells) engineered to express polynucleotides encoding the provided polynucleotides (including polynucleotides encoding CARs), 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 RNA (such as mRNA) transcribed from the construct after expression in cells. (i) Codon optimization

[0202] In some embodiments, the polynucleotide is modified by optimizing the codons used for expression in humans. In some aspects, codon optimization can be considered before and / or after steps for splice site identification and / or splice site elimination, and / or in each step of iterative steps for reducing RNA heterogeneity. Codon optimization typically involves balancing the percentage of selected codons with the abundance (e.g., publicly known abundance) of human transfer RNA, such that neither is overloaded or restricted. In some cases, this may be necessary or useful because most amino acids are encoded by more than one codon, and codon usage often varies from organism to organism. Differences in codon usage between the transfected or transduced gene or nucleic acid and the host cell can affect protein expression of the nucleic acid molecule. In some embodiments, to generate a codon-optimized nucleic acid sequence, codons are selected to balance those codons used in humans. Codon redundancy for amino acids ensures that different codons encode one amino acid. When selecting codons for substitution, the resulting mutation needs to be a silent mutation so that the codon change does not affect the amino acid sequence. Typically, the last nucleotide of a codon (e.g., at the third position) can remain unchanged without affecting the amino acid sequence. (ii) splice sites

[0203] This document provides polynucleotides in which one or more potential splice donor and / or splice acceptor sites have been identified, and nucleic acid sequences at or near one or more identified splice donor sites have been modified. In some embodiments, the resulting one or more modified nucleic acid sequences are then synthesized and used to transduce cells to test splicing, as indicated by RNA heterogeneity.

[0204] This document also provides polynucleotides (such as those encoding any antibodies, receptors (such as antigen receptors, such as chimeric antigen receptors), and / or GPRC5D-specific binding proteins provided herein) that have been modified or modified to reduce heterogeneity or contain one or more nucleic acid sequences observed herein (such as those obtained through the optimization methods described herein), resulting in improved characteristics of the polypeptide (such as CAR) compared to those containing different reference sequences or those that have not been modified. Such characteristics include improved RNA heterogeneity (such as RNA heterogeneity due to the presence of one or more splicing sites, such as one or more cryptic splicing sites) and / or improved expression and / or surface expression of the encoded protein, such as increased expression levels, uniformity, or consistency in cells engineered to express the polypeptide or in different therapeutic cell compositions.

[0205] Polynucleotides that have been modified to eliminate splicing sites (e.g., cryptic splicing sites) are also provided. Typically, genomic nucleic acid sequences naturally occurring in mammalian cells undergo co-transcriptional processing or immediate post-transcriptional processing, where nascent precursor messenger RNA (premRNA) transcribed from the genomic deoxyribonucleic acid (DNA) sequence is edited in some cases by splicing to remove introns, followed by ligation of exons in eukaryotic cells. The common sequences of splicing sites are known, while in some respects, the specific nucleotide information defining splicing sites can be complex and may not be readily apparent based on available methods. Cryptocryst splicing sites are splicing sites that are not predictable based on standard common sequences and are variablely activated. Therefore, alternative splicing of premRNA at cryptic splicing sites leads to heterogeneity in the expression of post-transcribed mRNA products in eukaryotic cells.

[0206] The polynucleotides used to express transgenes are typically constructed from nucleic acid sequences that do not contain introns, such as complementary DNA (cDNA) or portions thereof. Therefore, splicing of such sequences is not expected. However, the presence of cryptic splicing sites within the cDNA sequence can lead to unexpected or undesirable splicing responses and heterogeneity in the transcribed mRNA. This heterogeneity results in the translation of unintended protein products, such as truncated protein products with variable amino acid sequences exhibiting modified expression and / or activity.

[0207] In some embodiments, eliminating splice sites (such as cryptic splice sites) can improve or optimize the expression of transgenic products (such as peptides translated from transgenes, such as anti-GPRC5D CAR peptides). Splicing at cryptic splice sites encoding transgenes (such as GPRC5DCAR molecules) can lead to reduced protein expression (e.g., expression on the cell surface) and / or reduced function (e.g., reduced intracellular signaling). This document provides polynucleotides encoding anti-GPRC5D CAR proteins that have been optimized to reduce or eliminate cryptic splice sites. This document also provides polynucleotides encoding anti-GPRC5D CAR proteins that have been optimized for codon expression and / or contain one or more sequences (such as sequences identified by the methods or observations described herein regarding splice sites) and / or do not contain identified splice sites (such as any identified splice sites described herein). The provided polynucleotides include those that, when expressed under certain conditions and / or introduced into specific cell types (such as human T cells, like primary human T cells) and into cells and compositions and articles containing such polypeptides and / or exhibiting such properties, exhibit less than a certain level of RNA heterogeneity or splicing form. In some embodiments, the RNA heterogeneity of the transcribed RNA is reduced by more than or greater than about 10%, 15%, 20%, 25%, 30%, 40%, 50% or more compared to polynucleotides that are not modified to remove cryptic splice sites and / or are not modified through codon optimization. In some embodiments, the provided polynucleotides encoding anti-GPRC5D CAR exhibit RNA homogeneity of at least 70%, 75%, 80%, 85%, 90%, or 95% or higher for the transcribed RNA.

[0208] RNA heterogeneity can be determined by any of the various methods provided, described, or known herein. In some embodiments, RNA heterogeneity of transcribed nucleic acids is determined by amplifying the transcribed nucleic acids, such as by reverse transcriptase polymerase chain reaction (RT-PCR), followed by detecting one or more differences, such as size differences, in one or more amplification products. In some embodiments, RNA heterogeneity is determined based on the number of amplification products of different sizes or the proportion of amplification products of various different sizes. In some embodiments, RNA (such as total RNA or cytoplasmic polyadenylated RNA) is harvested from cells expressing the transgene to be optimized and amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using primers specific to the 5' untranslated region (5′ UTR) and primers specific to the 3' untranslated region (3′ UTR) or primers specific to a sequence within the transgene, wherein the 5' untranslated region in some cases corresponds to a portion of the promoter sequence in the expression vector, and the 3' untranslated region is downstream of the expressed transgene in the transcribed RNA sequence. In a particular embodiment, the transgene is amplified using 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). Skilled technicians can resolve RNA (such as messenger RNA) and analyze its heterogeneity using several methods. Non-limiting exemplary methods include agarose gel electrophoresis, chip-based capillary electrophoresis, analytical centrifugation, field fractionation, and chromatography (such as size exclusion chromatography or liquid chromatography).

[0209] In some aspects, the presence of potential cryptic splicing sites (splicing donor and / or acceptor sites) in transcripts (such as transgenic transcripts) can lead to RNA heterogeneity after transcript expression in cells. In some embodiments, one or more potential splicing sites that may be present in the transgenic transcript, unwanted, and / or generated from various potential sequences in the transgenic transcript after codon optimization and / or through mutations or errors in transcription are identified. In some aspects of the provided embodiments, the splicing donor site and the splicing acceptor site are identified independently. In some embodiments, the one or more splicing acceptor and / or donor sites are one or more classical, non-classical, and / or cryptic splicing acceptor and / or donor sites.

[0210] In some embodiments, one or more potential splicing sites (e.g., one or more classical, non-classical, and / or occult splice acceptor and / or donor sites or branching sites) in polynucleotides that may exhibit RNA heterogeneity (such as polynucleotides encoding transgenes, such as recombinant receptors) are identified and / or modified. Peptides having a reduced number of such splicing sites compared to such reference polynucleotides are also provided.

[0211] In some aspects, the identification of 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 a starting or reference sequence encoding a transgene (e.g., GPRC5D binding receptor, such as anti-GPRC5D) to a database, a gene synthesis supplier, or other sources capable of computationally or algorithmically comparing the starting or reference sequences 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, the sequence (e.g., a revised or modified nucleic acid sequence) undergoes one or more further evaluations to further assess splice site (e.g., cryptic splice site) removal using one or more other or additional splice site prediction tools.

[0212] In some respects, RNA heterogeneity may be a result of spliceosomal activity present in eukaryotic cells. In some respects, splicing typically occurs within a series of reactions catalyzed by the spliceosome. The common sequences of splice sites are known, while in others, the specific nucleotide information defining splice sites can be complex and may not be readily apparent based on available methods. Casually spliced ​​sites are splice sites that are variablely activated and cannot be predicted based on standard common sequences. Therefore, alternative splicing of pre-mRNA at casually spliced ​​sites leads to heterogeneity in the expression of post-transcribed mRNA products in eukaryotic cells. In some cases, within spliceosome introns, a donor site (typically at the 5' end of the intron), a branching site (near the 3' end of the intron), and a acceptor site (at the 3' end of the intron) are required for splicing events. Splice donor sites may contain a GU sequence at the 5' end of the intron and have a large, less conserved region. Splice acceptor sites at the 3' end of the intron may terminate with an AG sequence.

[0213] In some embodiments, one or more splice donor and / or acceptor sites are eliminated, such as potential splice donor and / or acceptor sites that may be involved in cryptic splicing events that could lead to unwanted RNA heterogeneity. In some embodiments, eliminating one or more splice sites includes modifying one or more nucleotides at, containing, or near the splice donor and / or acceptor site (e.g., by substitution or replacement), said splice donor and / or acceptor sites being candidates for removal. In some aspects, specific nucleotides at, containing, or near the splice site within a codon are modified (e.g., substituted or replaced). In some aspects, the modification (e.g., substitution or replacement) preserves or retains the amino acid encoded by the specific codon at said site while removing potential splice donor and / or acceptor sites.

[0214] In some embodiments, the codon at or near the splice site for modification comprises one or more codons that relate to one or both of the two nucleotides at the potential splice site (in some cases referred to as a "splice site codon"). When potential splicing is predicted to occur between two nucleotides in a codon, the codon is the only 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, then these two codons are splice site codons. For example, for a splice site predicted to be at the boundary of two codons, these two adjacent codons can be candidates for nucleotide modification. In some embodiments, the one or more codons include one splice site codon. In some embodiments, the one or more codons include 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, for example, one or both codons at the splice site are not modified when no synonymous codons for the splice site codon are available. In some embodiments, one or more nucleotides in a nearby codon may be modified if no available synonymous codons for a particular splice site codon are available. In some embodiments, the one or more codons to be modified include splice site codons, wherein the modification includes replacing one or two nucleotides at the splice site with one or more different nucleotides. In some embodiments, the splice donor site is eliminated by modifying one or two splice site codons, wherein the modification is not replacing one or two nucleotides at the splice site with different nucleotides, but rather modifying nearby nucleotides, such as a portion of a codon adjacent to the splice site. In some embodiments, the nearby or adjacent nucleotides that can be modified include nucleotides that modify a nearby or adjacent codon (such as codons one, two, three, four, five, six, seven, eight, nine, or ten within a codon upstream or downstream of the splice site codon).

[0215] 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 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, the one or more modifications are nucleotide substitutions or replacements of 1, 2, 3, 4, 5, 6, or 7 nucleotides. In some embodiments, the one or more modifications are at the junction of the splice donor site or 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 at the splice site junction of the splice acceptor and / or splice donor site. In some embodiments, a library of modified nucleic acid sequences can be generated with a reduced probability of cryptic splice sites. In some embodiments, splice donor sites and splice acceptor sites are evaluated as splice donor / acceptor pairs. In certain embodiments, the splice donor and acceptor sites are evaluated independently or separately, rather than as part of a splice donor / acceptor pair. In some embodiments, one or more predicted splice sites are not eliminated. In some embodiments, splice sites within the promoter region of the transcript, such as known or predicted splice sites, are not eliminated.

[0216] In some embodiments, one or more potential donor splicing sites are eliminated by modifying one or two splice site codons or one or more nearby or adjacent codons (e.g., if a synonymous codon is not available for the splice site codon). In some embodiments, one or more potential recipient splicing sites are eliminated by modifying one or two splice site codons or one or more nearby or adjacent codons (e.g., if a synonymous codon is not available for the splice site codon). In some embodiments, the nearby or adjacent codons to be modified include codons that are 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 from the splice site. In some embodiments, potential branching sites for splicing are removed or eliminated. In some aspects, nucleotides within codons at or near the branching site can be modified, for example, substituted or replaced, to eliminate covert splicing and / or reduce RNA heterogeneity. In some embodiments, the modification of the one or more nucleotides may involve the substitution or replacement of one of the nucleotides that may participate in splicing (e.g., at splice donor sites, splice acceptor sites, or splice branching sites) such that the amino acid encoded by the codon is preserved, and the nucleotide substitution or replacement does not alter the polypeptide sequence encoded by the polynucleotide. In some cases, the third position in the codon is more degenerate than the other two positions. Therefore, various synonymous codons can encode specific amino acids (see, for example, Section IA2.a above). In some embodiments, the modification includes replacing the codon with a synonymous codon used in the cell species in which the polynucleotide is introduced (e.g., humans). In some embodiments, the species is humans. In some embodiments, the one or more codons are replaced with the corresponding synonymous codon most frequently used in the species or a synonymous codon having a similar frequency of use to the corresponding codon (e.g., the closest frequency of use) (see, for example, Section IA2.a above).

[0217] In some aspects, the proposed modification can be further evaluated, for example, to assess whether the modification creates an unwanted or additional restriction site in the polynucleotide. In some aspects, additional restriction sites may not be needed, and other or different modifications can be considered (e.g., different nucleotide substitutions at the same codon or modifications at different positions or codons). 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, other or alternative modifications can be proposed. In some embodiments, after one or more iterations of the method, 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%.

[0218] In some embodiments, the provided polynucleotide encoding the anti-GPRC5D CAR or construct provided herein contains modifications to remove one or more splice donor and / or acceptor sites that may contribute to splicing events and / or reduced expression and / or increased RNA heterogeneity. In some embodiments, the provided polynucleotide is modified in one or more polynucleotides within a spacer region to eliminate or reduce splicing events. Potential splice donor and / or acceptor sites modified or excluded in the provided CAR are shown in SEQ ID NO: 194, 195, 196, 197, 198, or 199. In some embodiments, the modified nucleotides for reducing or eliminating potential splice and / or donor sites are shown in SEQ ID NO: 200, 201, 202, 203, 204, 205, or 206. In some embodiments, the provided polynucleotide encoding the anti-GPRC5D CAR or other CAR contains one or more nucleotide sequences shown in SEQ ID NO: 200, 201, 202, 203, 204, 205, or 206. In some embodiments, the provided anti-GPRC5D CAR contains a spacer sequence encoded by the nucleotide sequence shown in SEQ ID NO: 164. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 165. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 166. In some embodiments, the spacer is encoded by the nucleotide sequence shown in SEQ ID NO: 172. 3. Cells and cell preparation for genetic engineering

[0219] In some embodiments, the nucleic acid is heterologous, meaning it is not typically present in cells or samples obtained from cells, such as nucleic acids obtained from another organism or cell. For example, the nucleic acid is not typically found in engineered cells and / or the organism from which such cells originate. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acids not found in nature, including nucleic acids comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0220] The cells are typically 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 cells of innate or adaptive immunity, such as bone marrow or lymphocytes, including lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as pluripotent stem cells and multipotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are typically primary cells, such as those isolated directly from the subject and / or isolated from and frozen from the subject. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the entire T cell population, CD4+ cells, CD8+ cells, and their subsets, such as those defined by: function, activation state, maturity, potential for differentiation, expansion, recycling, localization and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion characteristics, and / or degree of differentiation. Regarding the subject to be treated, the cells can be allogeneic and / or autologous. The methods include off-the-shelf methods. In some respects, such as with existing technologies, cells are pluripotent and / or multipotent, such as stem cells, like induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from a subject, preparing, processing, culturing and / or engineering them, and reintroducing them into the same subject before or after cryopreservation.

[0221] T cell and / or CD4+ and / or CD8+ T cell subtypes and subsets include naive T cells (T0). N ) cells, effector T cells (T cells) EFF ), memory T cells and their subtypes (such as stem cell memory T cells (T15)). SCM ), central memory T (T CM ), effect memory T (T EMT cells include terminal differentiation effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated inertial 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.

[0222] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, such as bone marrow cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0223] In some embodiments, the cell includes one or more nucleic acids introduced through genetic engineering, and thereby expresses a recombinant product or a genetically engineered product of such nucleic acid. In some embodiments, the nucleic acid is heterologous, i.e., not normally present in the cell or in samples obtained from the cell, such as nucleic acids obtained from another organism or cell, for example, said nucleic acid is not typically found in the engineered cell and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as nucleic acids not found in nature, including nucleic acids comprising a chimeric combination of nucleic acids encoding various domains from a variety of different cell types.

[0224] In some embodiments, the preparation of engineered cells includes one or more culture and / or preparation steps. Cells used to introduce nucleic acids encoding transgenic receptors (such as CARs) can be isolated from a sample (such as a biological sample, for example, a biological sample obtained from or derived from a subject). In some embodiments, the subject from whom said cells are isolated is a subject suffering from a disease or condition, requiring cell therapy, or to whom cell therapy will be administered. In some embodiments, the subject is a person requiring a specific therapeutic intervention (such as adoptive cell therapy for which the cells are isolated, processed, and / or engineered).

[0225] Therefore, in some embodiments, the cells are primary cells, such as primary human cells. Samples include tissues, fluids, and other samples taken directly from the subject, as well as samples from one or more processing steps (such as separation, centrifugation, genetic engineering (e.g., transduction with a viral vector), washing, and / or incubation). Biological samples can be samples obtained directly from biological sources or processed samples. Biological samples include, but are not limited to, body fluids (such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat), tissue and organ samples, including processed samples derived therefrom.

[0226] In some respects, the sample from which cells are derived or isolated is blood or a blood-derived sample, or a product of apheresis or leukapheresis. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy material, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissues, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs and / or cells derived from them. In the case of cell therapy (e.g., adoptive cell therapy), samples include those from autologous and allogeneic sources.

[0227] In some embodiments, the cells are derived from cell lines, such as T cell lines. In some embodiments, the cells are obtained from xenogeneic sources, such as mice, rats, non-human primates, and pigs.

[0228] In some embodiments, cell isolation includes one or more preparative and / or non-affinity-based cell isolation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich desired components, or dissolve or remove cells sensitive to a specific reagent. In some examples, cells are isolated based on one or more properties, such as density, adhesion properties, size, sensitivity to a specific component, and / or resistance.

[0229] In some instances, cells are obtained from the subject's circulating blood via apheresis or leukoablation. In some aspects, the sample contains lymphocytes (including T cells, monocytes, granulocytes, and B cells), other nucleated leukocytes, erythrocytes, and / or platelets, and in some aspects, cells other than erythrocytes and platelets.

[0230] In some embodiments, blood cells collected from the subject are washed to remove plasma fractions, for example, and the cells are placed in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the wash solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is performed by a semi-automatic "flow-through" centrifuge (e.g., the Cobe2991 cell processor, Baxter), according to the manufacturer's instructions. In some aspects, the washing step is performed by tangential flow filtration (TFF), according to the manufacturer's instructions. In some embodiments, the cells are resuspended after washing in a variety of biocompatible buffers (e.g., calcium-free buffers). ++ / Mg ++ In some embodiments, components of the blood cell sample are removed and the cells are directly resuspended in the culture medium.

[0231] In some implementations, the method includes density-based cell separation methods, such as preparing leukocytes from peripheral blood by lysing red blood cells and centrifuging via Percoll or Ficoll gradient.

[0232] In some embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules (such as surface markers (e.g., surface proteins), intracellular markers, or nucleic acids) in the cells. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is based on affinity or immunoaffinity. For example, in some aspects, the separation includes separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers) in the cells, for example, by incubating with an antibody or binding partner that specifically binds to such a marker, followed typically by a washing step and separating cells that have bound the antibody or binding partner from those that have not.

[0233] Such separation steps can be based on positive selection (where cells that have already bound to the reagent are retained for further use) and / or negative selection (where cells that have not bound to the antibody or binding coupler are retained). In some cases, both fractions are retained for further use. In some respects, negative selection may be particularly useful in the absence of antibodies that can be used to specifically identify cell types in a heterogeneous population, making it best to separate based on markers expressed by cells other than the desired population.

[0234] The separation does not need to result in 100% enrichment or removal of a specific cell population or cells expressing a specific marker. For example, positive selection or enrichment for a specific type of cell (such as those expressing a marker) means increasing the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion for a specific type of cell (such as those expressing a marker) means reducing the number or percentage of such cells, but does not need to completely remove all such cells.

[0235] In some examples, multiple rounds of separation steps are performed, where fractions of positive or negative selection from one step undergo another separation step, such as subsequent positive or negative selection. In some examples, a single separation step can simultaneously deplete cells expressing multiple markers, such as by incubating cells with multiple antibodies or binding partners (each antibody or binding partner being specific for the marker targeted by negative selection). Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on various cell types.

[0236] For example, in some aspects, specific subpopulations of T cells are isolated using positive or negative selection techniques, such as cells that are positive for or express high levels of one or more surface markers, for example, CD28. + CD62L + CCR7 + CD27 + CD127 + CD4 + CD8 + CD45RA + and / or CD45RO + T cells.

[0237] For example, anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T cell expander) can be used to target CD3. + CD28 + T cells perform positive selection.

[0238] In some embodiments, separation is performed by enriching a specific cell population via positive selection or depleting a specific cell population via negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents, said antibodies or other binding agents being conjugated with markers expressed on the positively or negatively selected cells, respectively. + ) or expressed at a relatively high level (marker) 高 It specifically binds to one or more surface markers.

[0239] In some implementations, T cells are separated from PBMC samples by negatively selecting markers expressed on non-T cells (such as B cells, monocytes, or other leukocytes, such as CD14). In some aspects, CD4... + or CD8 + Select the step for separating CD4 + Helper T cells and CD8 + Cytotoxic T cells. These CD4+ cells can be identified through positive or negative selection based on markers expressed or expressed at relatively high levels in one or more naive, memory, and / or effector T cell subpopulations. + and CD8 + The group was further subdivided into multiple subgroups.

[0240] In some implementations, CD8 is further enriched or depleted. +Naïve, central memory, effector memory, and / or central memory stem cells within cells, such as through positive or negative selection based on surface antigens associated with the corresponding subpopulation. In some embodiments, targeting central memory T(T) cells is performed. CM Enrichment of T cells to increase efficacy, such as to improve long-term survival, expansion, and / or transplantation after administration, is particularly robust in some respects in this subpopulation. See Terakura et al. (2012) Blood, 1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some implementations, T cells are enriched. CM CD8 + T cells and CD4 + The T-cell combination further enhances efficacy.

[0241] In the implementation plan, memory T cells are present in CD8. + CD62L of peripheral blood lymphocytes + and CD62L - Two subsets. PBMC can be targeted at CD62L. - CD8 + and / or CD62L + CD8 + Fractions can be enriched or depleted, for example, using anti-CD8 and anti-CD62L antibodies.

[0242] In some implementations, central memory T(T) CM Cell enrichment is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, the enrichment is based on negative selection of cells expressing or highly expressing CD45RA and / or granzyme B. In some aspects, enrichment of T cells is performed by depletion of cells expressing CD4, CD14, and CD45RA and positive selection or enrichment of cells expressing CD62L. CM CD8 cells + Group separation. In one aspect, central memory T(T CM Cell enrichment begins with a negative cell fraction selected based on CD4 expression, which undergoes negative selection based on CD14 and CD45RA expression and positive selection based on CD62L expression. In some respects, this selection occurs simultaneously, while in others it occurs sequentially in any order. In some respects, in the preparation of CD8... + The same CD4 expression-based selection steps used in cell populations or subpopulations are also used to generate CD4. +Cell populations or subpopulations, such that both positive and negative fractions from CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more other positive or negative selection steps.

[0243] In specific cases, PBMC samples or other white blood cell samples are subjected to CD4. + Cell selection is performed, retaining both negative and positive fractions. The negative fraction is then selected based on the expression of CD14 and CD45RA or CD19, and the positive fraction is selected based on marker characteristics of central memory T cells (such as CD62L or CCR7), wherein the positive and negative selections are performed in any order.

[0244] By identifying cell populations with cell surface antigens, CD4 + T helper cells were sorted into naive, central memory, and effector cells. CD4 + Lymphocytes can be obtained using standard methods. In some implementations, naïve CD4 cells are used. + T lymphocytes are CD45RO - CD45RA + CD62L + CD4 + T cells. In some implementations, central memory CD4... + The cells are CD62L + And CD45RO + In some implementations, the effect is CD4. + The cells are CD62L - And CD45RO - .

[0245] In one example, to enrich CD4 through negative selection +Cellular monoclonal antibody mixtures typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or conjugate is bound to a solid support or matrix (such as magnetic or paramagnetic beads) to allow for the separation of cells for positive and / or negative selection. For example, in some embodiments, immunomagnetic (or affinity magnetic) separation techniques are used to separate or isolate cells and cell populations (reviewed in Methods in Molecular Medicine, Vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, pp. 17–25, edited by SA Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0246] In some aspects, a sample or composition of cells to be separated is incubated with small magnetizable or magnetically responsive materials, such as magnetically responsive particles or microparticles, like paramagnetic beads (e.g., Dynabeads or MACS beads). Typically, the magnetically responsive material (e.g., particles) is attached directly or indirectly to a binding partner (e.g., an antibody) that specifically binds to a molecule (e.g., a surface marker) present on a cell, multiple cells, or cell population to be separated (e.g., to select for negative or positive cells).

[0247] In some embodiments, the magnetic particles or beads comprise a magnetically responsive material that binds to a specific binding member, such as an antibody or other binding partner. Many well-known magnetically responsive materials are used in magnetic separation methods. Suitable magnetic particles include those described in the following documents: Molday, U.S. Patent No. 4,452,773 and European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal-sized particles are other examples, such as those described in the following documents: Owen, U.S. Patent No. 4,795,698 and Liberti et al., U.S. Patent No. 5,200,084.

[0248] Incubation is typically carried out under conditions in which antibodies or binding couplers, or molecules (such as secondary antibodies or other reagents) specifically bound to such antibodies or binding couplers attached to magnetic particles or beads, specifically bind to cell surface molecules (if present on cells within the sample).

[0249] In some aspects, the sample is placed in a magnetic field, and those cells with magnetically responsive or magnetizable particles attached are attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted by the magnet are retained; for negative selection, cells not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, wherein positive and negative fractions are retained for further processing or undergo additional separation steps.

[0250] In some embodiments, the magnetically responsive particles are coated with a primary antibody or other binding chaperone, a secondary antibody, a lectin, an enzyme, or streptavidin. In some embodiments, the magnetic particles attach to cells by coating with one or more label-specific primary antibodies. In some embodiments, the cells are labeled with a primary antibody or binding chaperone instead of beads, and then the magnetic particles coated with a cell-type-specific secondary antibody or other binding chaperone (e.g., streptavidin) are added. In some embodiments, the streptavidin-coated magnetic particles are used in combination with a biotinylated primary or secondary antibody.

[0251] In some embodiments, the magnetically responsive particles remain attached to the cells, which are subsequently incubated, cultured, and / or engineered; in some aspects, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competitive unlabeled antibodies and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0252] In some embodiments, affinity-based selection is performed via magnetically activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California). Magnetic activated cell sorting (MACS) systems are capable of high-purity selection of cells with attached magnetized particles. In some embodiments, MACS operates in a manner where non-target and target species are eluted sequentially after an external magnetic field is applied. That is, cells attached to the magnetized particles remain in place, while unattached species are eluted. Then, after the first elution step is completed, the species trapped in the magnetic field and prevented from elution are released in a way that allows them to be eluted and recovered. In some embodiments, the non-target cells are tagged and depleted from a heterogeneous cell population.

[0253] In some embodiments, a system, apparatus, or device is used for separation or splitting, performing one or more of the separation, cell preparation, splitting, processing, incubation, culture, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, for example to minimize errors, user error, and / or contamination. In one example, the system is as described in International Patent Application Publication No. WO 2009 / 072003 or US 20110003380 A1.

[0254] In some implementations, the system or device performs one or more (e.g., all) of the separation, processing, engineering, and formulation steps in an integrated or stand-alone system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer programs communicating with the system or device, allowing a user to program, control, evaluate outcomes, and / or adjust various aspects of the processing, separation, engineering, and formulation steps.

[0255] In some respects, the CliniMACS system (Miltenyi Biotec) is used for separation and / or other steps, for example, for the automated separation of cells at a clinical scale in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various clamp valves. In some respects, a computer controls all components of the instrument and instructs the system to perform repetitive procedures in a standardized sequence. In some respects, the magnetic separation unit includes a movable permanent magnet and a support for selecting columns. The peristaltic pump controls the flow rate throughout the tubing assembly and, together with the clamp valves, ensures controlled flow of buffer solution and continuous cell suspension within the system.

[0256] In some aspects, the CliniMACS system uses antibody-conjugated magnetizable particles supplied in a sterile, pyrogen-free solution. In some embodiments, after labeling cells with the magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing assembly, which in turn connects to a bag containing buffer and a cell collection bag. The tubing assembly consists of pre-assembled sterile tubing (including a pre-column and a separation column) and is for single use only. Upon initiation of the separation procedure, the system automatically applies the cell sample to the separation column. Labeled cells remain in the column, while unlabeled cells are removed through a series of washing steps. In some embodiments, the cell population used for use with the methods described herein is unlabeled and not retained in the column. In some embodiments, the cell population used for use with the methods described herein is labeled and retained in the column. In some embodiments, the cell population used for use with the methods described herein is eluted from the column after removal of the magnetic field and collected in a cell collection bag.

[0257] In some implementations, the CliniMACS Prodigy system (Miltenyi Biotec) is used for separation and / or other steps. In some aspects, the CliniMACS Prodigy system is equipped with a cell handling unit that allows for automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by identifying macroscopic layers of the source cell products. For example, peripheral blood is automatically separated into erythrocytes, leukocytes, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber that enables cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports allow for aseptic removal and replenishment of culture medium, and cells can be monitored using an integrated microscope. See, for example, Klebanoff et al. (2012) J Immunother. 35(9): 651-660; Terakura et al. (2012) Blood. 1:72–82; and Wang et al. (2012) J Immunother. 35(9):689-701.

[0258] In some embodiments, the cell population described herein is collected and enriched (or depleted) via flow cytometry, wherein the fluid stream carries cells stained against multiple cell surface markers. In some embodiments, the cell population described herein is collected and enriched (or depleted) via preparative stage (FACS) sorting. In some embodiments, the cell population described herein is collected and enriched (or depleted) by using a microelectromechanical system (MEMS) chip in conjunction with a FACS-based detection system (see, for example, WO 2010 / 033140; Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1(5):355-376). In both cases, cells can be labeled with multiple markers, thereby allowing the separation of well-defined subsets of T cells with high purity.

[0259] In some implementations, antibodies or binding partners are labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation can be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on binding to one or more cell surface markers or other binding partners is performed in a fluidic flow, such as via fluorescence-activated cell sorting (FACS) (including preparative-grade FACS) and / or microelectromechanical systems (MEMS) chips, for example, in combination with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0260] In some embodiments, the preparation method includes the step of freezing (e.g., cryopreservation) cells before or after isolation, incubation, and / or engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes from the cell population and, to some extent, monocytes. In some embodiments, cells are suspended in a freezing solution, for example, after a washing step, to remove plasma and platelets. In some aspects, any of a variety of known freezing solutions and parameters can be used. One example involves using PBS containing 20% ​​DMSO and 8% human serum albumin (HSA), or other suitable cell freezing medium. This is then diluted 1:1 with the medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. The cells are then typically frozen to -80°C at a rate of 1°C / min and stored in the gas phase of a liquid nitrogen tank.

[0261] In some embodiments, cells are incubated and / or cultured prior to or in conjunction with genetic engineering. The incubation step may include culturing, nurturing, stimulating, activating, and / or proliferating. Incubation and / or engineering may be performed in culture vessels, such as units, chambers, wells, columns, tubes, tube sets, valves, vials, culture dishes, bags, or other containers used for culturing or nurturing cells. In some embodiments, the composition or cells are incubated under stimulating conditions or in the presence of a stimulant. Such conditions include those designed to: induce cell proliferation, expansion, activation, and / or survival in a population; simulate antigen exposure; and / or induce cells for genetic engineering (e.g., for the introduction of recombinant antigen receptors).

[0262] The conditions may include one or more of the following: specific culture medium, temperature, oxygen content, carbon dioxide content, time, and agents (e.g., nutrients, amino acids, antibiotics, ions and / or stimulating factors (such as cytokines, chemokines, antigens, binding couplers, fusion proteins, recombinant soluble receptors and any other agents designed to activate cells)).

[0263] In some embodiments, the stimulating condition or stimulant includes one or more agents (e.g., ligands) capable of activating or stimulating intracellular signaling domains of the TCR complex. In some aspects, the agent opens or initiates the TCR / CD3 intracellular signaling cascade in T cells. Such agents may include antibodies, such as antibodies specific to the TCR, e.g., anti-CD3 antibodies. In some embodiments, the stimulating condition includes one or more agents, such as ligands, capable of stimulating co-stimulatory receptors, e.g., anti-CD28. In some embodiments, such agents and / or ligands may bind to a solid support (e.g., beads) and / or one or more cytokines. Optionally, the amplification method may further include the step of adding anti-CD3 and / or anti-CD28 antibodies to a culture medium (e.g., at a concentration of at least about 0.5 ng / mL). In some embodiments, the stimulant includes IL-2, IL-15, and / or IL-7. In some aspects, the IL-2 concentration is at least about 10 units / mL.

[0264] In some respects, incubation is carried out using a variety of techniques, such as those described in the following literature: U.S. Patent No. 6,040,177 to Riddell et al.; Klebanoff et al. (2012) J Immunother. 35(9):651-660; Terakura et al. (2012) Blood. 1:72-82; and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0265] In some embodiments, T cells are expanded by adding feeder cells (such as non-dividing peripheral blood mononuclear cells (PBMCs)) to the culture starting composition (e.g., such that for each T lymphocyte in the initial population to be expanded, the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells); and incubating the culture (e.g., for a duration sufficient to expand the number of said T cells). In some aspects, the non-dividing feeder cells may comprise γ-irradiated PBMC feeder cells. In some embodiments, PBMCs are irradiated with γ rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, feeder cells are added to the culture medium prior to the addition of the T cell population.

[0266] In some embodiments, stimulation conditions include a temperature suitable for the growth of human T lymphocytes, such as at least about 25 degrees Celsius, typically at least about 30 degrees Celsius, and typically at or about 37 degrees Celsius. Optionally, incubation may also include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCLs) as feeder cells. LCLs can be irradiated with gamma rays in the range of about 6,000 to 10,000 rads. In some aspects, LCL feeder cells are provided in any suitable amount, such as an LCL feeder cell to naïve T lymphocyte ratio of at least about 10:1.

[0267] In the implementation scheme, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen-specific T lymphocytes with an antigen. For example, antigen-specific T cell lines or clones against cytomegalovirus antigens can be generated by isolating T cells from an infected subject and stimulating said cells in vitro with the same antigen. 4. Compositions and formulations

[0268] In some embodiments, the dosage of cells in cell therapies (such as T-cell therapies, which comprise cells engineered with recombinant antigen receptors (e.g., CARs)) is provided as a composition or formulation (such as a pharmaceutical composition or formulation). Such compositions may be used according to the provided methods and / or used in conjunction with the provided products or compositions.

[0269] In some implementations, cell therapies (such as engineered T cells, e.g., CAR T cells) are formulated using pharmaceutically acceptable carriers. In some aspects, the choice of carrier is determined in part by the specific cells or drug and / or by the method of administration. Therefore, a variety of suitable formulations exist. For example, the pharmaceutical composition may contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. Preservatives or mixtures thereof are typically present in an amount from about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in: Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed. 1980). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10). (Residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0270] In some aspects, the composition includes a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing an administerable pharmaceutical composition are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0271] The formulation may include an aqueous solution. The formulation or composition may also contain more than one active ingredient that can be used to treat a specific indication, disease, or condition with cells or pharmaceutical agents, wherein the individual activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Therefore, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vincristine, etc.

[0272] In some embodiments, the pharmaceutical composition contains a number of cells effective in treating the disease or condition (e.g., a therapeutically effective amount or a preventatively effective amount). In some embodiments, therapeutic efficacy is monitored by periodically evaluating the treated subject. For repeated administration over several days or longer, depending on the condition, treatment is repeated until the desired suppression of disease symptoms occurs. However, other dosing regimens may be useful and can be determined. The required dose can be delivered by a single bolus injection, by multiple bolus injections, or by continuous infusion.

[0273] The cells can be administered using standard application techniques, formulations, and / or devices. Formulations and devices, such as syringes and vials, are provided for storing and administering the composition. Regarding the cells, administration can be autologous or allogeneic. For example, immune-response cells or progenitor cells can be obtained from a subject and administered to the same subject or different compatible subjects. Peripheral blood-derived immune-response cells or their progeny (e.g., in vivo, ex vivo, or in vitro derived) can be administered via local injection (including catheter administration), systemic injection, local injection, intravenous injection, or parenteral administration. When administering therapeutic compositions (e.g., pharmaceutical compositions containing genetically modified immune-response cells), they are typically formulated into injectable, unit-dose forms (solutions, suspensions, emulsions).

[0274] Preparations include those intended for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the drug or cell population is administered parenterally. As used herein, the term "parenterectomy" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the drug or cell population is administered to the subject via peripheral systemic delivery, such as intravenous, intraperitoneal, or subcutaneous injection.

[0275] In some embodiments, the composition is provided as a sterile liquid formulation, such as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, which may be buffered to a selected pH in some respects. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are slightly easier to administer, particularly by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a particular tissue. Liquid or viscous compositions may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0276] Sterile injectable solutions can be prepared by incorporating cells into a solvent, for example, by mixing with a suitable carrier, diluent, or excipient (such as sterile water, physiological saline, glucose, dextrose, etc.).

[0277] Preparations intended for internal administration are typically sterile. Sterility can be readily achieved, for example, by filtration through a sterile filter membrane. 5. Dosage and administration of anti-GPRC5D CAR T-cell therapy

[0278] The provided embodiments include methods of administration and uses (e.g., therapeutic and preventative uses) of anti-GPRC5D CAR T-cell therapy. Such methods and uses include, for example, treatments and uses involving administering to a subject suffering from a disease, condition, or disorder associated with GPRC5D, such as a disease, condition, or disorder associated with GPRC5D expression and / or in which cells or tissues express (e.g., specifically express) GPRC5D. In some embodiments, the molecule, cell, and / or composition is administered in an effective amount to achieve treatment of the disease or disorder. This document provides the use of recombinant receptors (e.g., CARs) and cells (e.g., engineered cells) in such methods and treatments, as well as in the preparation of pharmaceutical agents for performing such treatments. In some embodiments, the methods are performed by administering a binding molecule or cell, or a composition containing said binding molecule or cell, to a subject who has, has, or is suspected of having a disease or condition. In some embodiments, the methods thereby treat the subject's disease or condition or disorder. This document also provides for the use of any of the described compositions (such as the pharmaceutical compositions provided herein) in the treatment of diseases or disorders associated with GPRC5D, such as in a treatment regimen.

[0279] Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the methods and compositions provided. For example, adoptive T-cell therapy methods are described in: for example, U.S. Patent Application Publication No. 2003 / 0170238 by Gruenberg et al.; U.S. Patent No. 4,690,915 by Rosenberg; Rosenberg (2011) Nat RevClin Oncol. 8(10):577-85. See also, for example, Themeli et al. (2013) Nat Biotechnol. 31(10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.

[0280] In some embodiments, the method includes adoptive cell therapy, whereby genetically engineered cells expressing a provided recombinant receptor containing a GPRC5D binding molecule (e.g., a CAR containing an anti-GPRC5D antibody or its antigen-binding fragment) are administered to a subject. This administration can promote cell activation (e.g., T cell activation) in a GPRC5D-targeted manner, thereby targeting and destroying diseased or disordered cells.

[0281] In some embodiments, the method includes administering cells or a composition containing said cells to a subject, tissue, or cell, such as a subject, tissue, or cell that has, is at risk of having, or is suspected of having a disease, condition, or disorder. In some embodiments, cells, populations, and compositions are administered, for example, via adoptive cell therapy such as adoptive T-cell therapy, to a subject suffering from a specific disease or condition to be treated. In some embodiments, cells or compositions are administered to a subject, such as a subject who has or is at risk of having a disease or condition. In some aspects, the method thereby treats the disease or condition (e.g., improves one or more symptoms of the disease or condition), such as by reducing the tumor burden in cancers expressing GPRC5D.

[0282] In some implementations, cell therapy (e.g., adoptive cell therapy, such as adoptive T-cell therapy) is performed via autologous transfer, wherein cells are isolated and / or otherwise prepared from a subject to receive the cell therapy or from a sample derived from that subject. Thus, in some aspects, cells are derived from a subject requiring treatment (e.g., a patient), and the cells are administered to the same subject after isolation and processing.

[0283] In some embodiments, cell therapy (e.g., adoptive cell therapy, such as adoptive T-cell therapy) is performed via allogeneic transfer, wherein cells are isolated and / or otherwise prepared from a subject other than the subject to receive or ultimately receive the cell therapy (e.g., a first subject). In such embodiments, the cells are then administered to a different subject of the same species, such as a second subject. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0284] In some embodiments, the subject to which the cells, cell populations, or composition are administered is a primate, such as a human. In some embodiments, the subject to which the cells, cell populations, or composition are administered is a non-human primate. In some embodiments, the non-human primate is a monkey (e.g., a cynomolgus monkey) or an ape. The subject can be male or female and can be of any suitable age, including infants, young children, adolescents, adults, and elderly subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent (e.g., a mouse, rat, etc.). In some examples, the patient or subject is an empirical animal model used for disease, adoptive cell therapy, and / or for evaluating toxic outcomes such as cytokine release syndrome (CRS).

[0285] GPRC5D binding receptors (e.g., CARs) and cells expressing GPRC5D binding receptors can be administered by any suitable means, such as injection, including intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intra-anterior chamber injection, subconjunctival injection, sub-Tenon injection, retroocular injection, periocular injection, or posterior juxtascleral delivery. In some embodiments, they are administered via parenteral, intrapulmonary, and intranasal administration, as well as (if local treatment requires) intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration can depend in part on whether the administration is short-term or long-term. Various dosing schedules include, but are not limited to, single or multiple administrations at different time points, bolus administration, and pulsatile infusion.

[0286] For the prevention or treatment of disease, the appropriate dosage of the binding molecule, recombinant receptor, or cell may depend on the type of disease to be treated, the type of binding molecule or recombinant receptor, the severity and course of the disease, whether the administration of the binding molecule or recombinant receptor is for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the recombinant receptor or cell, and the attending physician's decision. In some embodiments, the composition and molecules and cells are suitably administered to the patient once or in a series of treatments.

[0287] In some embodiments, the dose and / or frequency of administration are determined based on efficacy and / or response. In some embodiments, efficacy is determined by assessing disease status. Exemplary methods for assessing disease status include: measuring M protein in biological fluids (such as blood and / or urine) by electrophoresis and immunofixation; quantification of sFLC (κ and λ) in blood; skeletal examination; and imaging of a subject with extramedullary disease by positron emission tomography (PET) / computed tomography (CT). In some embodiments, disease status may be assessed by bone marrow examination.

[0288] In some embodiments, the dose and / or frequency of administration is determined by the expansion and persistence of the recombinant receptor or cells in the blood and / or bone marrow. In some embodiments, the dose and / or frequency of administration is determined based on the antitumor activity of the recombinant receptor or engineered cells. In some embodiments, antitumor activity is determined by the overall response rate (ORR) and / or the International Myeloma Working Group (IMWG) Harmonized Response Criteria (see Kumar et al. (2016) Lancet Oncol 17(8):e328-346). In some embodiments, minimal residual disease (MRD) assessment is used to evaluate the response. In some embodiments, MRD can be assessed by methods such as flow cytometry and high-throughput sequencing (e.g., deep sequencing). In some embodiments, the response is evaluated based on the duration of the response following administration of the recombinant receptor or cells. In some examples, the dose and / or frequency of administration may be based on toxicity. In some embodiments, the dose and / or frequency may be determined based on the health-related quality of life (HRQoL) of the subject administering the recombinant receptor and / or cells. In some implementations, the dosage and / or frequency of administration can be varied based on any of the above criteria, i.e., increased or decreased.

[0289] In some embodiments, the method includes administering a dose of the engineered cells or a composition containing a dose of the engineered cells. In some embodiments, the engineered cells or a composition containing engineered cells may be used in a treatment regimen that includes administering a dose of the engineered cells or a composition containing a dose of the engineered cells. In some embodiments, the dose may contain, for example, a specific number or range of recombinant receptor-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), any number of such cells as described herein. In some embodiments, a composition containing a dose of the cells may be administered. In some aspects, the number, amount, or proportion of CAR-expressing cells in a cell population or cell composition can be assessed by detecting the binding of an alternative marker (e.g., by flow cytometry or other means) or by detecting the binding of a labeled molecule (such as a labeled antigen) that can specifically bind to a binding molecule or receptor provided herein.

[0290] In some implementations, in the context of genetically engineered cells containing binding molecules or recombinant receptors, the subject is given cells ranging from approximately one million to approximately 100 billion cells and / or the number of cells per kilogram of body weight, such as, for example, approximately one million to approximately 50 billion cells (e.g., approximately 5 million cells, approximately 10 million, approximately 12.5 million, approximately 15 million, approximately 20 million, approximately 25 million, approximately 500 million, approximately 1 billion, approximately 5 billion, approximately 20 billion, approximately 30 billion, approximately 40 billion cells, or a range defined by any two of the foregoing values), such as approximately 10 million to approximately 100 billion cells (e.g., approximately 10 million, approximately 12.5 million, approximately 15 million, 20 million, approximately 25 million, approximately 30 million, approximately 1 ... 10,000 cells, approximately 40 million cells, approximately 50 million cells, approximately 60 million cells, approximately 70 million cells, approximately 80 million cells, approximately 90 million cells, approximately 10 billion cells, approximately 25 billion cells, approximately 50 billion cells, approximately 75 billion cells, approximately 90 billion cells, or a range defined by any two of the foregoing values), and in some cases approximately 100 million cells to approximately 50 billion cells (e.g., approximately 120 million cells, approximately 150 million cells, approximately 250 million cells, approximately 300 million cells, approximately 350 million cells, approximately 450 million cells, approximately 500 million cells, approximately 600 million cells, approximately 650 million cells, approximately 800 million cells, approximately 900 million cells, approximately 1 billion cells, approximately 1.2 billion cells, or approximately 3 billion cells) or any value between these ranges and / or these ranges per kilogram of body weight. Similarly, the dosage can vary depending on the disease or impairment and / or patient- and / or other treatment-specific properties. In some implementations, the number of cells is the number of such cells as living cells.

[0291] In some embodiments, the provided method implements a flat dose, such as the total number of CAR+ cells, CAR+CD8+ T cells, and / or the total number of CAR+CD4+ T cells, as if administering a precise or fixed dose of one or more such cell types to each of a group of subjects being treated (including subjects with variable weight). Therefore, the provided method includes a method in which the cell dose is a flat or fixed dose of cells, such that the cell dose is not related to or based on the subject's body surface area or weight. In some embodiments, this method can minimize or reduce the chance of administering too many cells to the subject, which could increase the risk of toxic outcomes associated with CAR-T cell administration.

[0292] In some implementations, such as when the subject is a human, the dose contains more than about 1 x 10⁻⁶. 6The number of total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) is less than approximately 2 x 10⁻⁶. 9 Total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), for example, at approximately 2.5 x 102 7 From approximately 1.2 x 10 9 Within the range of such cells, such as 2.5 x 10 7 5 x 10 7 7.5 x 10 7 1.5 x 10 8 3 x 10 8 4.5 x 10 8 8 x 10 8 Or 1.2 x 10 9 The total number of such cells, or any two of the aforementioned values ​​within a range. In some embodiments, such as when the subject is human, the dose contains more than about 1 x 103 cells. 6 The number of total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) is less than approximately 2 x 10⁻⁶. 9 Total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), for example, at approximately 1.0 x 10⁻⁶ cells / year. 7 From approximately 1.2 x 10 9 Within the range of such cells, such as 1.0 x 10 7 1.25 x 10 7 1.5 x 10 7 2.0 x 10 7 2.5 x 10 7 5 x 10 7 7.5 x 10 7 1.5 x 10 8 2.25 x 10 8 3 x 10 8 4.5 x 10 8 6.0 x 10 8 8 x 10 8 Or 1.2 x 10 9 The total number of such cells, or any two of the aforementioned values ​​within a range. In some embodiments, such as when the subject is human, the dose contains more than about 1 x 103 cells. 6 The number of total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) is less than approximately 2 x 10⁻⁶. 9 Total CAR-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), for example, at approximately 1.0 x 10⁻⁶ cells / year. 7To approximately 6.5 x 10 8 One such cell, approximately 1.5 x 10 7 From approximately 6.0 x 10 8 One such cell, approximately 1.5 x 10 7 To approximately 6.5 x 10 8 One such cell, approximately 2.5 x 10 7 From approximately 6.0 x 10 8 One such cell, or approximately 5.0 x 10 7 From approximately 6.0 x 10 8 The number of such cells is within a range. In some implementations, the number of cells is the number of such cells as living cells.

[0293] In some implementations, the dose of genetically engineered cells is contained in approximately 2.5 x 10⁻⁶ ml. 7 The number of CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs) is approximately 1.2 x 10^12. 9 Between CAR-expressing T cells, total T cells, or total PBMCs, the number is approximately 5.0 x 10⁻⁶. 7 Each CAR-expressing T cell has approximately 4.5 x 10⁻⁶ cells. 8 Between CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), the number is approximately 7.0 x 10⁻⁶. 7 Each CAR-expressing T cell is approximately 2.0 x 10^12 cells. 8 Between CAR-expressing T cells, total T cells, or total PBMCs (each including end values). In some embodiments, the number is relative to the total number of CD3+ or CD8+ cells, and in some cases, it is also relative to CAR-expressing (e.g., CAR+) cells. In some embodiments, the dose comprises from or from about 2.5 x 10 7 Up to or approximately 1.2 x 10 9 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 5.0 x 10 7 Up to or approximately 4.5 x 10 8 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, or from approximately 7 x 10 7 Up to or approximately 2.0 x 10 8 Each contains either a CD3+ or CD8+ total T cell or a CD3+ or CD8+ CAR-expressing cell, each containing a terminal value. In some embodiments, the number of cells is the number of such cells as live cells. In some embodiments, the number of cells is the number of CD3+ CAR-expressing cells as live cells.

[0294] In some implementations, the dose of genetically engineered cells is contained in a volume of approximately 1.0 x 10⁻⁶. 7 The number of CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs) is approximately 1.2 x 10^12. 9 Between CAR-expressing T cells, total T cells, or total PBMCs, the number is approximately 2.0 x 10⁻⁶. 7 Each CAR-expressing T cell has approximately 4.5 x 10⁻⁶ cells. 8 Between CAR-expressing T cells, total T cells, or total peripheral blood mononuclear cells (PBMCs), the number is approximately 7.0 x 10⁻⁶. 7 Each CAR-expressing T cell is approximately 2.0 x 10^12 cells. 8 Between CAR-expressing T cells, total T cells, or total PBMCs (each including end values). In some embodiments, the number is relative to the total number of CD3+ or CD8+ cells, and in some cases, it is also relative to CAR-expressing (e.g., CAR+) cells. In some embodiments, the dose comprises the following number of cells: from or from about 1.0 x 10 7 Up to or approximately 1.2 x 10 9 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 1.5 x 103 7 Up to or approximately 1.2 x 10 9 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 2.5 x 10 7 Up to or approximately 1.2 x 10 9个 CD3+ or CD8+ total T cells or CD3+ or CD8+ CAR-expressing cells, from or from approximately 1.5 x 10-1 7 Up to or approximately 8.0 x 10 8 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 2.5 x 10 7 Up to or approximately 8.0 x 10 8 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 1.5 x 103 7 Up to or approximately 6.0 x 10 8 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 2.5 x 10 7 Up to or approximately 6.0 x 10 8 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 5.0 x 10 7Up to or approximately 6.0 x 10 8 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, from or from approximately 5.0 x 10 7 Up to or approximately 4.5 x 10 8 One CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell, or from approximately 7.0 x 103 7 Up to or approximately 2.0 x 10 8 Each CD3+ or CD8+ total T cell or CD3+ or CD8+ CAR-expressing cell contains end values. In some embodiments, the number of cells is the number of such cells as live cells.

[0295] In some implementations, the dose of genetically engineered cells comprises 2.5 x 10 7 CAR-expressing cells. In some embodiments, the dose of genetically engineered cells contains 7.5 x 10⁻⁶ CAR-expressing cells. 7 CAR-expressing cells. In some embodiments, the dose of genetically engineered cells contains 7.5 x 10⁻⁶ CAR-expressing cells. 7 CAR-expressing T cells. In some implementations, the dose of genetically engineered cells contains 1.5 x 10⁻⁶ CAR-expressing T cells. 8 One CAR-expressing cell. In some embodiments, the dose of genetically engineered cells contains 1.5 x 10⁻⁶ CAR-expressing cells. 8 CAR-expressing T cells. In some implementations, the number of cells is the number of such cells as living cells.

[0296] In some embodiments, the dose of T cells includes CD4+ T cells, CD8+ T cells, or CD4+ and CD8+ T cells.

[0297] In some implementations, for example, when the subject is a human, the dose (including doses containing CD4+ and CD8+ T cells) comprises CD8+ T cells in a dose of or approximately 1 x 102 6 With or approximately 2 x 10 9 Total CAR expression in CD8+ cells between 100 cells, for example, in cells with a total expression of approximately 5 x 100 cells. 7 At most or approximately 4.5 x 10 8 Within the range of such cells, such as approximately 2.5 x 103 7 , or approximately 5 x 10 7 It is approximately 7.5 x 10 7 It is approximately 1.5 x 10 8 , or approximately 3 x 10 8 It is approximately 4.5 x 108 , or approximately 8 x 10 8 or approximately 1.2 x 10 9 The total number of such cells, or any two of the aforementioned values ​​within a range. In some embodiments, the number of cells is the number of such cells as living cells.

[0298] In the context of adoptive cell therapy, the administration of a given “dose” of CAR-expressing cells includes administering a given amount or number of cells in a single composition and / or a single, uninterrupted administration (e.g., by a single injection or continuous infusion), and also includes administering a given amount or number of cells in a fractionated dose or multiple compositions provided in a plurality of individual compositions or infusions over a specified time period, such as not exceeding 3 days. Thus, in some cases, the dose is a single or continuous administration of a specified number of cells, given or initiated at a single time point. However, in some cases, the dose is administered in multiple injections or infusions over a time period not exceeding three days, such as once daily for three days or for two days, or by multiple infusions over a single day.

[0299] Therefore, in some aspects, the dose of cells is administered as a single pharmaceutical composition. In some embodiments, the dose of cells is administered as a combination of multiple compositions that collectively contain said dose of cells.

[0300] In some embodiments, the term "fractionated dose" refers to a dose that is divided such that it is administered over a period of more than one day. This type of administration is covered in the methods of the present invention and is considered a single dose.

[0301] Therefore, the cellular dose can be administered as a fractionated dose, such as a fractionated dose administered over time. For example, in some embodiments, the dose may be administered to the subject over 2 or 3 days. Exemplary methods for fractionated dosing include administering 25% of the dose on the first day and the remaining 75% on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% on the second day. In some aspects, 10% of the dose may be administered on the first day, 30% on the second day, and 60% on the third day. In some embodiments, the fractionated dose may not exceed 3 days.

[0302] In some embodiments, the dose of cells can be administered by applying multiple compositions or solutions (such as first and second, optionally more), each composition or solution containing a portion of the dose of cells. In some aspects, multiple compositions, each containing a different cell population and / or cell subtype, may optionally be administered separately or independently over a period of time. For example, the cell population or subtype may each comprise CD8. + and CD4 +T cells, and / or CD8 enriched respectively + and enriched CD4 + Groups, for example, each individually comprising CD4 cells genetically engineered to express the recombinant receptor. + and / or CD8 + T cells. In some embodiments, dosage administration includes administering a first composition comprising a dose of CD8. + T cells or a certain dose of CD4 + T cells, and administration of a second composition comprising the stated dose of CD4. + T cells and CD8 + Another type is found in T cells.

[0303] In some embodiments, dosage administration (e.g., administration of the multiple cell compositions) involves administering the cell composition alone. In some aspects, the individual administration is performed simultaneously or sequentially in any order. In a particular embodiment, individual administration is performed sequentially by administering the following in any order: a first composition, the first composition containing CD8. + T cell dose or CD4 + A dose of T cells, and a second composition comprising the dose of CD4. + T cells and CD8 + Another type of T cell. In some embodiments, the dose comprises a first composition and a second composition, and the first composition and the second composition are administered within 48 hours of each other, such as within 36 hours of each other or within 24 hours of each other. In some embodiments, the first composition and the second composition are administered 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart. In some embodiments, the start of administration of the first composition and the start of administration of the second composition are performed within 2 hours, 1 hour, or 30 minutes, or within 15 minutes, 10 minutes, or 5 minutes. In some embodiments, the start and / or completion of administration of the first composition and the completion and / or start of administration of the second composition are performed within 2 hours, 1 hour, or 30 minutes, or within 15 minutes, 10 minutes, or 5 minutes. In some embodiments, the first composition and the second composition are administered less than 2 hours apart.

[0304] In some compositions, the first composition (e.g., the dose of the first composition) contains CD4. + T cells. In some compositions, the first composition (e.g., the dose of the first composition) contains CD8. +T cells. In some embodiments, the first composition is administered before the second composition. In a particular embodiment, CD8+ T cells are administered before CD4+ T cells.

[0305] In some embodiments, the cell dose or composition comprises a defined or targeted ratio of CD4 expressing a recombinant receptor (e.g., CAR). + Cells and CD8 cells expressing recombinant receptors (e.g., CAR) + Cells and / or defined or targeted ratios of CD4 + Cells and CD8 + Cells, the ratio of which is optionally about 1:1 or between about 1:3 and about 3:1, such as about 1:1. In some respects, different cell populations (such as CD4) with a target or desired ratio. + CD8 + Ratio or CAR + CD4 + :CAR + CD8 + The administration of a composition or dose at a ratio, such as 1:1, involves administering a cell composition containing one population followed by the administration of a separate cell composition containing another population, wherein the administration is performed at or approximately at a target or desired ratio. In some respects, administering a dose or composition of cells at a determined ratio results in improved expansion, persistence, and / or antitumor activity of T-cell therapy.

[0306] In some embodiments, the applied cells are immune cells engineered to express GPRC5D-binding recombinant receptors (e.g., CAR). In some embodiments, the immune cells are T cells. In some embodiments, the applied cells are CD4+ T cells. In some embodiments, the applied cells are CD8+ T cells. In some embodiments, the applied cells are a combination of CD4+ and CD8+ T cells, such as CAR T cells. In some examples, the ratio of CD4+ cells to CD8+ cells (CD4:CD8) is 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1.

[0307] In some implementations, for example, when the subject is a human, the dose (including doses containing CD4+ and CD8+ T cells) comprises CD8+ T cells in a dose of or approximately 1 x 102 6 With or approximately 2 x 10 9The total number of recombinant receptors (e.g., CARs) expressed in CD8+ cells, for example, in cells with a total of approximately 1 x 10⁻⁶ cells, is approximately 1 x 10⁻⁶ cells. 7 At most or approximately 4.5 x 10 8 Within the range of such cells, such as approximately 1.0 x 103 7 It is approximately 1.25 x 10 7 It is approximately 1.5 x 10 7 , or approximately 2.0 x 10 7 It is approximately 2.5 x 10 7 , or approximately 5 x 10 7 It is approximately 7.5 x 10 7 It is approximately 1.5 x 10 8 , or approximately 3 x 10 8 It is approximately 4.5 x 10 8 It is approximately 6.0 x 10 8 , or approximately 8 x 10 8 or approximately 1.2 x 10 9 The total number of such cells, or any two of the aforementioned values ​​within the range.

[0308] In some implementations, an exemplary dose comprises approximately 5.0 x 10 7 7.5 x 10 7 1.5 x 10 8 3.0 x 10 8 Or 4.5 x 10 8 CAR-expressing T cells. In some embodiments, exemplary doses are in the following range: 1.0 x 10 7 With 1.5 x 10 7 Between, 1.25 x 10 7 With 2.5 x 10 7 Between, 2.0 x 10 7 With 3.5 x 10 7 Between, 3.0 x 10 7 With 4.5 x 10 7 Between, 4.0 x 10 7 With 7.5 x 10 7 Between, 5.0 x 10 7 With 2.25 x 10 8 Between, 1.5 x 10 8 With 4.5 x 10 8 Between, or 3.0 x 10 8 With 6.0 x 10 8Between CAR-expressing T cells. In some embodiments, an exemplary dose includes approximately 1.0 x 10 7 1.25 x 10 7 1.5 x 10 7 2.0 x 10 7 2.0 x 10 7 2.5 x 10 7 3.0 x 10 7 3.5 x 10 7 4.0 x 10 7 4.5 x 10 7 5.0 x 10...

Claims

1. A method for treating relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) Administering CAR-T cell therapy comprising a dose of engineered cells to a subject with RRMM, said engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D); and (2) The subject was given a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3.

2. The method of claim 1, wherein the bispecific antibody is administered according to a dosing regimen that begins no earlier than 60 days after administration of the CAR T-cell therapy.

3. The method according to claim 1 or 2, wherein the bispecific antibody comprises at least one Fab fragment of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody.

4. The method according to claim 1 or 2, wherein the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody.

5. The method according to any one of claims 1-4, wherein at least one fixed dose of the bispecific antibody is administered in an amount between 1 mg and 50 mg.

6. The method according to any one of claims 2-5, wherein the administration regimen of the bispecific antibody is a cyclic regimen comprising more than one treatment cycle.

7. The method of claim 6, wherein each treatment cycle is a 28-day cycle.

8. The method according to any one of claims 2-7, wherein the dosing regimen of the bispecific antibody includes a first treatment cycle (C1), the first treatment cycle being an escalating dosing regimen comprising one or more initial doses and one or more maintenance doses, wherein the maintenance dose is greater than the initial dose.

9. The method of claim 8, wherein C1 further comprises one or more intermediate doses, wherein the intermediate doses are greater than the initial dose and less than the maintenance dose.

10. A method for treating relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) Administering CAR-T cell therapy comprising a dose of engineered cells to a subject with RRMM, said engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D); and (2) The subject was administered a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3 using the following administration regimen, which was initiated after administration of the CAR-T cells; The dosing regimen for the bispecific antibody includes a cyclical regimen comprising more than one treatment cycle, wherein each treatment cycle is a 28-day treatment cycle, and wherein the first treatment cycle (C1) is an escalating dosing regimen, the escalating dosing regimen comprising: (i) The initial phase, wherein one or more initial doses of the bispecific antibody are administered to the subject; (ii) An intermediate phase, wherein one or more intermediate doses of the bispecific antibody are administered to the subject, wherein the intermediate dose is greater than the initial dose; and (iii) Maintenance phase, wherein the subject is administered one or more maintenance doses of the bispecific antibody, wherein each maintenance dose is greater than the intermediate dose.

11. The method of claim 10, wherein the bispecific antibody comprises at least one Fab fragment of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody.

12. The method of claim 10, wherein the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody.

13. The method according to any one of claims 10-12, wherein the bispecific antibody is administered 28 days to 6 months after the subject is given the CAR-T cell therapy.

14. The method according to any one of claims 10-13, wherein the bispecific antibody is administered no earlier than 60 days after the CAR-T cell therapy.

15. The method according to any one of claims 1-14, wherein the bispecific agent is administered at or about 60 days after the CAR-T cell therapy.

16. The method according to any one of claims 8-15, wherein the initial phase comprises a single initial dose.

17. The method according to any one of claims 8-16, wherein the starting dose is about 1.0 mg to 6 mg.

18. The method according to any one of claims 8-17, wherein the starting dose is about 3 mg.

19. The method according to any one of claims 9-18, wherein the intermediate stage comprises a single intermediate dose.

20. The method according to any one of claims 9-19, wherein the intermediate dose is about 4.5 mg to 10 mg.

21. The method according to any one of claims 9-20, wherein the intermediate dose is about 6.0 mg.

22. The method according to any one of claims 8-21, wherein the maintenance dose is about 6 mg to about 50 mg.

23. The method according to any one of claims 8-22, wherein the maintenance dose is about 25.0 mg to 35.0 mg.

24. The method according to any one of claims 8-23, wherein the maintenance dose is about 30 mg.

25. The method according to any one of claims 8-21, wherein the maintenance dose is about 6 mg to 18 mg.

26. The method according to any one of claims 8-21 and 25, wherein the maintenance dose is about 10 mg.

27. The method according to any one of claims 8-26, wherein the one or more initial doses are administered during the first week of C1.

28. The method according to any one of claims 8-27, wherein a single starting dose is administered on day 1 (D1) of C1.

29. The method according to any one of claims 9-28, wherein the one or more intermediate doses are administered during the first week of C1.

30. The method according to any one of claims 9-29, wherein a single intermediate dose is administered on one of days 3-6 of C1.

31. The method according to any one of claims 9-30, wherein the single intermediate dose is administered on day 4 (D4) of C1.

32. The method according to any one of claims 8-28, wherein in C1, the one or more maintenance doses are administered once a week after the first week.

33. The method according to any one of claims 8-32, wherein in C1, the one or more maintenance doses are administered on day 8 (D8), day 15 (D15), and day 22 (D22).

34. The method according to any one of claims 8-33, wherein in C1, the bispecific antibody is administered at about 3 mg on day 1, about 6 mg on day 4, and about 30 mg on days 8, 15, and 22.

35. The method according to any one of claims 8-34, wherein the dosing regimen further comprises two or more consecutive treatment cycles, each treatment cycle comprising a weekly dosing of the maintenance dose (Q1W).

36. The method of claim 35, wherein the dosing regimen comprises 2-4 treatment cycles performed once a week.

37. The method according to any one of claims 8-36, wherein the dosing regimen includes a second treatment cycle (C2), the second treatment cycle including once-weekly dosing of the maintenance dose.

38. The method according to any one of claims 8-36, wherein the dosing regimen comprises a second treatment cycle (C2) and a third treatment cycle (C3), wherein each of C2 and C3 is characterized by once-weekly dosing of the maintenance dose.

39. The method of claims 37 and 38, wherein the maintenance dose is administered once weekly on days 1, 8, 15, and 22.

40. The method of claim 38, wherein C2 comprises administering the bispecific antibody at about 30 mg on days 1, 8, 15 and 22, and C3 comprises administering the bispecific antibody at about 30 mg on days 1, 8, 15 and 22.

41. The method according to any one of claims 35-40, wherein after two or more consecutive treatment cycles each comprising once-weekly administration (Q1W), the dosing regimen comprises one or more additional treatment cycles characterized by a reduced frequency of administration of the maintenance dose of the bispecific antibody.

42. The method of claim 41, wherein the one or more additional treatment cycles comprise dosing every other week (Q2W), dosing once a month (Q4W), or a combination thereof.

43. The method of claim 41 or claim 42, wherein the one or more additional treatment cycles comprise one or more treatment cycles of administering the maintenance dose at Q2W.

44. The method of claim 41, wherein the dosing regimen comprises 2-6 treatment cycles of administering the maintenance dose at Q2W.

45. The method according to any one of claims 42-44, wherein for each treatment cycle of Q2W administration, the maintenance dose of Q2W administration is administered on day 1 and day 15 of each cycle.

46. ​​The method according to any one of claims 41-45, wherein the one or more additional treatment cycles further comprise one or more treatment cycles of administering the maintenance dose Q2W after one or more treatment cycles of administering the maintenance dose Q4W.

47. The method according to any one of claims 41-46, wherein the dosing regimen comprises 4-10 treatment cycles of administering the maintenance dose at Q4W.

48. The method according to any one of claims 41-47, wherein for each treatment cycle of Q4W administration, the maintenance dose of Q4W administration is administered on day 1 of each cycle.

49. The method according to any one of claims 1-48, wherein the bispecific antibody is administered to the subject for a maximum of 12 treatment cycles.

50. A method for treating relapsed and / or refractory multiple myeloma (RRMM), the method comprising: (1) CAR-T cell therapy comprising a dose of engineered cells comprising T cells expressing chimeric antigen receptor (CAR) that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D) in a subject with RRMM. (2) The subject is given a bispecific antibody that binds to B cell maturation antigen (BCMA) and CD3, wherein the bispecific antibody comprises two Fab fragments of an anti-BCMA antibody and one Fab fragment of an anti-CD3 antibody, and wherein the bispecific antibody is given in a dosing regimen that begins no earlier than 60 days after the administration of the CAR T cells, and wherein the dosing regimen is characterized by 12 treatment cycles, each of which is 28 days. in: (i) The first treatment cycle of the bispecific antibody administration includes administration of 3 mg on day 1, 6 mg on day 4, and 30 mg on days 8, 15 and 22; (ii) The second and third treatment cycles of the bispecific antibody each consist of administering 30 mg on days 1, 8, 15 and 22; (iii) The fourth, fifth and sixth treatment cycles of the bispecific antibody each consist of administering 30 mg on day 1 and day 15; (iv) Each of the 7th to 12th treatment cycles of the bispecific antibody includes the administration of 30 mg on day 1.

51. The method according to any one of claims 1-50, wherein the bispecific antibody is administered subcutaneously.

52. The method according to any one of claims 1-51, wherein the bispecific antibody is a trivalent bispecific antibody comprising two Fab fragments of an anti-BCMA antibody, one Fab fragment of an anti-CD3 antibody, and an Fc portion, optionally wherein the bispecific antibody is in the form of BCMA Fab-Fc-CD3 Fab-BCMA Fab.

53. The method according to any one of claims 3-52, wherein the anti-BCMA Fab comprises: (a) Heavy chain variable regions (VH) containing the VH sequence shown in SEQ ID NO: 210 and light chain variable regions (VL) containing the VL sequence shown in SEQ ID NO:

216. (b) VH containing the VH sequence shown in SEQ ID NO: 210 and VL containing the VL sequence shown in SEQ ID NO: 217; (c) VH containing the VH sequence shown in SEQ ID NO: 210 and VL containing the VL sequence shown in SEQ ID NO: 218; (d) VH containing the VH sequence shown in SEQ ID NO: 211 and VL containing the VL sequence shown in SEQ ID NO: 216; (e) VH containing the VH sequence shown in SEQ ID NO: 212 and VL containing the VL sequence shown in SEQ ID NO: 216; (f) VH containing CDR1, CDR2, and CDR3 of the VH sequence shown in SEQ ID NO: 213, and VL containing CDR1, CDR2, and CDR3 of the VL sequence shown in SEQ ID NO: 216; or (g) VH containing CDR1, CDR2 and CDR3 of the VH sequence shown in SEQ ID NO: 214 and VL containing CDR1, CDR2 and CDR3 of the VL sequence shown in SEQ ID NO:

215.

54. The method according to any one of claims 1-53, wherein the anti-BCMA Fab comprises: (a) A VH containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 225, 226 and 221 respectively; and a VL containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 227, 228 and 224 respectively; (b) A VH containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 225, 226 and 221 respectively; and a VL containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 229, 230 and 224 respectively; (c) A VH containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 225, 226 and 221 respectively; and a VL containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 231, 232 and 224 respectively; (d) A VH containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 233, 234 and 221 respectively; and a VL containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 235, 236 and 237 respectively; (e) A VH containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 238, 239 and 221 respectively; and a VL containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 235, 236 and 237 respectively; (f) A VH containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 240, 241, and 221, respectively; and a VL containing the CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 235, 236, and 237, respectively; or (g) A VH containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 219, 220 and 221 respectively; and a VL containing the CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 222, 223 and 224 respectively.

55. The method according to any one of claims 3-54, wherein the anti-BCMA Fab comprises a VH containing the VH sequence shown in SEQ ID NO: 210 and a VL containing the VL sequence shown in SEQ ID NO:

218.

56. The method according to any one of claims 3-55, wherein the anti-BCMA Fab comprises a VH containing CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 225, 226 and 221 respectively; and a VL containing CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 231, 232 and 224 respectively.

57. The method according to any one of claims 3-56, wherein the anti-CD3 antibody or its antigen-binding fragment comprises a VH containing the VH sequence shown in SEQ ID NO: 242 and a VL containing the VL sequence shown in SEQ ID NO:

243.

58. The method according to any one of claims 3-57, wherein the anti-CD3 antibody or its antigen-binding fragment comprises a VH containing CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 244, 245 and 246, respectively; and a VL containing CDR1, CDR2 and CDR3 sequences as shown in SEQ ID NO: 247, 248 and 249, respectively.

59. The method according to any one of claims 1-58, wherein the bispecific antibody comprises a combination of the heavy and light chains of the sequences shown in SEQ ID NO:250, SEQ ID NO:251, and SEQ ID NO:252, and two copies of the sequence shown in SEQ ID NO:

253.

60. The method according to any one of claims 1-59, wherein the bispecific antibody is anoxtabumab.

61. The method according to any one of claims 1-60, wherein the CAR comprises (1) an extracellular antigen-binding domain that specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D); (2) an immunoglobulin hinge spacer of at least 125 amino acids in length; (3) a transmembrane domain; and (4) an intracellular signal transduction region comprising a cytoplasmic signal transduction domain of a CD3-ζ (CD3ζ) chain and a co-stimulatory signal transduction region; The extracellular antigen-binding domain contains V H District and V L District, of which: The V H The region contains the amino acid sequence shown in SEQ ID NO: 1 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% identity with SEQ ID NO: 1; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 2 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% identity with SEQ ID NO: 2; The V H The region contains the amino acid sequence shown in SEQ ID NO: 1 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% identity with SEQ ID NO: 1; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 116 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% identity with SEQ ID NO: 116; The V H The region contains the amino acid sequence shown in SEQ ID NO: 3 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% identity with SEQ ID NO: 3; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 4 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% identity with SEQ ID NO: 4; The V H The region contains the amino acid sequence shown in SEQ ID NO: 3 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% identity with SEQ ID NO: 3; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 117 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% identity with SEQ ID NO: 117; The V H The region contains the amino acid sequence shown in SEQ ID NO: 5 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% identity with SEQ ID NO: 5; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 6 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% identity with SEQ ID NO: 6; The V H The region contains the amino acid sequence shown in SEQ ID NO: 5 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% identity with SEQ ID NO: 5; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 118 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% identity with SEQ ID NO: 118; The V H The 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% identity with SEQ ID NO: 7; and the V L The region contains 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% identity with SEQ ID NO: 8; The V H The 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% identity with SEQ ID NO: 7; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 119 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% identity with SEQ ID NO: 119; The V H The region contains the amino acid sequence shown in SEQ ID NO: 9 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% identity with SEQ ID NO: 9; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 10 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% identity with SEQ ID NO: 10; The V H The region contains the amino acid sequence shown in SEQ ID NO: 9 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% identity with SEQ ID NO: 9; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 120 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% identity with SEQ ID NO: 120; The V H The region contains the amino acid sequence shown in SEQ ID NO: 11 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% identity with SEQ ID NO: 11; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 12 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% identity with SEQ ID NO: 12; The V H The region contains the amino acid sequence shown in SEQ ID NO: 11 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% identity with SEQ ID NO: 11; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 121 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% identity with SEQ ID NO: 121; The V H The region contains the amino acid sequence shown in SEQ ID NO: 13 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% identity with SEQ ID NO: 13; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 14 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% identity with SEQ ID NO: 14; or The V H The region contains the amino acid sequence shown in SEQ ID NO: 13 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% identity with SEQ ID NO: 13; and the V L The region contains the amino acid sequence shown in SEQ ID NO: 122 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% identity with SEQ ID NO:

122.

62. The method of claim 61, wherein the extracellular antigen-binding domain comprises V H District and V L District, of which: The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences of SEQ ID NO: 20, 21, and 17, and the V L The region contains amino acid sequences of SEQ ID NO: 25, 26 and 27, respectively; The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 35, 36, and 32, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 40, 41, and 42; The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 50, 51, and 47, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 55, 56, and 57; The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 65, 66, and 62, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 70, 71, and 72; The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively containing the amino acid sequences SEQ ID NO: 80, 81, and 77, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 85, 86, and 87; The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 80, 94, and 91, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 97, 98, and 99; The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 107, 108, and 104, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, respectively, containing the amino acid sequences of SEQ ID NO: 112, 26, and 113; or The V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 109, 110, and 111, and the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 114, 29, and 123.

63. The method of claim 61 or claim 62, wherein the extracellular antigen-binding domain comprises V H District and V L District, of which: The V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 1 and 2, or amino acid sequences as shown in SEQ ID NO: 1 and 116, respectively; The V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 3 and 4, or amino acid sequences as shown in SEQ ID NO: 3 and 117, respectively; The V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 5 and 6, or amino acid sequences as shown in SEQ ID NO: 5 and 118, respectively; The V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 7 and 8, or amino acid sequences as shown in SEQ ID NO: 7 and 119, respectively; The V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 9 and 10, or amino acid sequences as shown in SEQ ID NO: 9 and 120, respectively; The V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 11 and 12, or amino acid sequences as shown in SEQ ID NO: 11 and 121, respectively; or The V H The area and the V L The region contains amino acid sequences as shown in SEQ ID NO: 13 and 14, or amino acid sequences as shown in SEQ ID NO: 13 and 122, respectively.

64. The method according to any one of claims 61-63, wherein the extracellular antigen-binding domain comprises V H District and V L The area, the V H The region comprises CDR-H1, CDR-H2, and CDR-H3, respectively, containing the amino acid sequences SEQ ID NO: 65, 66, and 62, wherein the V L The region contains CDR-L1, CDR-L2, and CDR-L3, which respectively contain the amino acid sequences of SEQ ID NO: 70, 71, and 72.

65. The method according to any one of claims 61-64, wherein: The V H The 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% identity with SEQ ID NO: 7; And the V L The region contains the amino acid sequence shown in SEQ ID NO: 119 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% identity with SEQ ID NO:

119.

66. The method according to any one of claims 61-65, wherein: The V H The region contains the amino acid sequence shown in SEQ ID NO: 7, and the V L The region contains the amino acid sequence shown in SEQ ID NO:

119.

67. The method according to any one of claims 61-66, wherein the extracellular antigen-binding domain is a single-chain antibody fragment, and the single-chain antibody fragment is or comprises a single-chain variable fragment (scFv).

68. The method according to any one of claims 61-67, wherein the V H The area and the V L The areas are connected by flexible joints.

69. The method of claim 68, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 127).

70. The method according to any one of claims 61-69, wherein the V H The region is located in the V L The carboxyl terminus of the region.

71. The method according to any one of claims 61-70, wherein the extracellular antigen-binding domain comprises an amino acid sequence selected from SEQ ID NO: 131, 133, 137 and 141 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 an amino acid sequence selected from SEQ ID NO: 131, 133, 137 and 141.

72. The method according to any one of claims 61-71, wherein the extracellular antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:

137.

73. The method according to any one of claims 61-72, wherein the spacer comprises sequences of the immunoglobulin hinge region, the CH2 region, and the CH3 region.

74. The method according to any one of claims 61-73, wherein the spacer comprises an IgG4 / 2 chimeric hinge region or a modified IgG4 hinge region containing at least one amino acid substitution compared to the human IgG4 hinge; IgG2 / 4 chimeric C H Region 2; and IgG4 C H Zone 3.

75. The method according to any one of claims 61-74, wherein the spacer is or comprises (i) the sequence shown in SEQ ID NO: 162; (ii) a functional variant of SEQ ID NO: 162 having 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: 162; or (iii) a continuous portion of (i) or (ii) having a length of at least 125 amino acids.

76. The method according to any one of claims 61-75, wherein the spacer is encoded by the nucleotide sequence shown in SEQ ID NO:

164.

77. The method according to any one of claims 61-76, wherein the cytoplasmic signal transduction domain of CD3ζ comprises the amino acid sequence shown in SEQ ID NO: 176 or an amino acid 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:

176.

78. The method according to any one of claims 61-77, wherein the co-stimulatory signal transduction region comprises an intracellular signal transduction domain of CD28, 4-1BB, or ICOS, or a signal transduction portion thereof.

79. The chimeric antigen receptor according to any one of claims 61-78, wherein the co-stimulatory signal transduction region comprises an intracellular signal transduction domain of 4-1BB.

80. The method according to any one of claims 61-79, wherein the co-stimulatory signal transduction region is or comprises the amino acid sequence shown in SEQ ID NO: 179 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 sequence shown in SEQ ID NO:

179.

81. The method according to any one of claims 61-80, wherein the transmembrane domain is or comprises a transmembrane domain from CD4, CD28 or CD8.

82. The method according to any one of claims 61-81, wherein the transmembrane domain is or comprises a transmembrane domain derived from CD28.

83. The method according to any one of claims 61-82, wherein the CAR comprises: (1) An extracellular antigen-binding domain, wherein the extracellular antigen-binding domain specifically binds to human G protein-coupled receptor class C5 member D (GPRC5D), wherein the extracellular antigen-binding domain comprises: (i) A variable heavy chain (V) containing 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. H ) area; and (ii) A variable light chain (V) containing 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:

119. L )district; (2) A spacer comprising an IgG4 / 2 chimeric hinge or a modified IgG4 hinge; an IgG2 / 4 chimeric C H Region 2; and IgG4 C H In region 3, the spacer may optionally be approximately 228 amino acids in length; (3) Transmembrane domains from human CD28; and (4) Intracellular signal transduction region, wherein the intracellular signal transduction region comprises a cytoplasmic signal transduction domain of the CD3-ζ (CD3ζ) chain and an intracellular signal transduction domain of 4-1BB.

84. The method according to claim 83, wherein: The extracellular antigen-binding domain includes the V shown in SEQ ID NO:

7. H The amino acid sequence of the region and the V shown in SEQ ID NO:119 L The amino acid sequence of the region.

85. The method according to claim 83 or 84, wherein the extracellular antigen-binding domain comprises the scFv shown in SEQ ID NO:

137.

86. The method according to any one of claims 83-85, wherein: The spacer is or comprises (i) the sequence shown in SEQ ID NO: 162; (ii) a functional variant of SEQ ID NO: 162 having at least or at least 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: 162; or (iii) a continuous portion of (i) or (ii) having a length of at least 125 amino acids; or The spacer is encoded by the nucleotide sequence shown in SEQ ID NO:

164.

87. The method according to any one of claims 61-86, wherein the transmembrane domain is or comprises the amino acid sequence shown in SEQ ID NO: 173 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:

173.

88. The method according to any one of claims 61-87, wherein the intracellular signal transduction region comprises (a) a cytoplasmic signal transduction domain of a CD3-ζ (CD3ζ) chain, the cytoplasmic signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 176 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: 176, and (b) an intracellular signal transduction domain of 4-1BB, the intracellular signal transduction domain comprising the amino acid sequence shown in SEQ ID NO: 179 or an amino acid sequence having at least or at least about 99% sequence identity with SEQ ID NO:

176. The sequence shown in 179 has amino acid sequence identity of 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%.

89. The method according to any one of claims 61-87, wherein the intracellular signal transduction region is or comprises the sequences shown in SEQ ID NO: 176 and SEQ ID NO:

179.

90. The method according to any one of claims 1-89, wherein the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO: 183 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:

183.

91. The method according to any one of claims 1-89, wherein the chimeric antigen receptor is encoded by the nucleotide sequence shown in SEQ ID NO:182 or a nucleotide 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:

182.

92. The method according to any one of claims 1-91, wherein the dose of engineered cells comprises the following number of CAR-expressing T cells: at about 1.0 x 10 7 CAR-expressing T cells and 1.2 x 10 9 Between CAR-expressing T cells, at approximately 1.0 x 10 7 CAR-expressing T cells with 6.5 x 10 8 Between CAR-expressing T cells, at approximately 1.5 x 10 7 CAR-expressing T cells with 6.5 x 10 8 Between CAR-expressing T cells, at approximately 1.5 x 10 7 CAR-expressing T cells with 6.0 x 10 8 Between CAR-expressing T cells, at approximately 2.5 x 10 7 CAR-expressing T cells with 6.0 x 10 8 Between CAR-expressing T cells, at approximately 5.0 x 10 7 CAR-expressing T cells with 6.0 x 10 8 Between CAR-expressing T cells, at approximately 1.25 x 10 7 CAR-expressing T cells and 1.2 x 10 9 Between CAR-expressing T cells, at approximately 1.5 x 10 7 CAR-expressing T cells and 1.2 x 10 9 Between CAR-expressing T cells, at approximately 5.0 x 10 7 CAR-expressing T cells and 4.5 x 10 8 Between CAR-expressing T cells, or at approximately 1.5 x 10 8 CAR-expressing T cells and 3.0 x 10 8 Between each CAR-expressing T cell, there is an end value.

93. The method according to any one of claims 1-92, wherein the dose of engineered cells comprises or is about 1.5 x 10⁻⁶. 7 It is approximately 2.5 x 10 7 It is approximately 5.0 x 10 7 It is approximately 7.5 x 10 7 It is approximately 1.5 x 10 8 It is approximately 2.25 x 10 8 , or approximately 3.0 x 10 8 It is approximately 4.5 x 10 8 It is approximately 6.0 x 10 8 It is approximately 8.0 x 10 8 or approximately 1.2 x 10 9 One CAR-expressing T cell.

94. The method according to any one of claims 1-93, wherein the dose of engineered cells comprises or is about 7.5 x 10⁻⁶. 7 One CAR-expressing T cell.

95. The method according to any one of claims 1-94, wherein the method further comprises administering lymphocyte depletion chemotherapy to the subject prior to administering the CAR-T cell therapy.

96. The method of claim 95, wherein the lymphocyte clearance therapy is completed approximately 7 days prior to the initiation of administration of the dose of the CAR-expressing T cells.

97. The method of claim 95 or 96, wherein the administration of the lymphocyte clearance therapy is completed approximately 2 to 7 days prior to the initiation of administration of the dose of engineered T cells.

98. The method according to any one of claims 95-97, wherein the lymphocyte clearance therapy comprises administration of fludarabine and / or cyclophosphamide.

99. The method according to any one of claims 95-98, wherein the lymphocyte clearance therapy comprises the administration of fludarabine and cyclophosphamide.

100. The method according to any one of claims 95-99, wherein the lymphocyte clearance therapy comprises daily administration of 200-400 mg / m². 2 Cyclophosphamide, containing end-values, can optionally be administered daily at a dose of approximately 300 mg / m². 2 Cyclophosphamide.

101. The method according to any one of claims 95-99, wherein the lymphocyte clearance therapy comprises daily administration of 20-40 mg / m² or approximately. 2 Fludarabine, including the end value, can optionally be administered daily at a dose of approximately 30 mg / m². 2 Fluoradabine.

102. The method according to any one of claims 95-101, wherein the lymphocyte clearance therapy comprises administering fludarabine and cyclophosphamide for 2-4 days, optionally 3 days.

103. The method according to any one of claims 95-98, wherein the lymphocyte clearance therapy comprises administration of bendamustine.

104. The method according to any one of claims 95-98 and 103, wherein the lymphocyte clearance therapy comprises daily administration of 50-130 mg / m² or approximately 2 Bendamustine, containing end-point, can optionally be administered daily at a dose of approximately 90 mg / m². 2 Bendamustine.

105. The method according to any one of claims 95-98, 103 and 104, wherein the lymphocyte clearance therapy comprises administering bendamustine for 1-3 days, optionally 2 days.

106. The method according to any one of claims 1-105, wherein the dosing regimen of the bispecific antibody combined with the CAR-T cell therapy prevents the subject from developing toxicities characterized by one or more of the following: (i) Any CRS of grade 4 and any CRS that has not regressed to grade < 2 within 72 hours; (ii) Grade 3 ICANS or neurotoxicity (NT) for any duration; (iii) Grade 3 or higher toxicity affecting vital organs (e.g., heart, lungs) for any duration; (iv) All other Grade 3 toxicities that do not resolve to ≤ Grade 2 within 72 hours and cannot be attributed to an underlying disease or lymphocyte clearance chemotherapy, excluding non-CRS toxicities of the liver, wherein liver transaminase levels are ≥ 3x the upper limit of normal (ULN), accompanied by an increase in total bilirubin to > 2x ULN, but without an initial finding of cholestasis (e.g., elevated serum alkaline phosphatase), and no other cause can be found to explain the synchronous increase in transaminase and total bilirubin, such as hepatitis A, B or C, pre-existing liver disease or acute liver disease or another drug that can cause the observed liver injury; (v) Grade 3 aspartate aminotransferase (AST) and / or alanine aminotransferase (ALT) elevation lasting ≤ 14 days or Grade 4 AST and / or ALT elevation lasting ≤ 7 days; (vi) Isolated grade 3 or 4 asymptomatic laboratory electrolyte abnormalities that are not directly related to major organ toxicity and occur during certain treatments, which resolve to grade ≤ 2 within ≤ 7 days with or without intervention; (vii) Grade 4 thrombocytopenia that has not resolved to grade 3 or lower within 14 days or is accompanied by clinically significant bleeding, or Grade 3 thrombocytopenia accompanied by clinically significant bleeding; (viii) Despite the use of granulocyte colony-stimulating factor (G-CSF), the neutropenia did not resolve to grade 4 or lower within 14 days; (ix) Any Grade 5 toxicity not associated with disease progression; (x) Any macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH); or (xi) Any thrombotic event of grade 3 or higher.

107. The method according to any one of claims 1-106, wherein the subject does not develop serious toxicity.

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