Humanized nanobody targeting BCMA and use thereof

Abstract

Provided are humanized nanobody targeting BCMA and a use thereof. The nanobody is capable of binding to B-cell maturation antigen (BCMA), and the isolated antigen-binding protein comprises at least one CDR in an antibody heavy chain variable region (VH). Provided are a chimeric antigen receptor, a fusion protein, a cell and a nucleic acid molecule comprising the antigen-binding protein, and a use thereof in the treatment of tumors.

Description

A humanized nanobody targeting BCMA and its application Technical Field

[0001] This application relates to the field of biomedicine, specifically to an anti-BCMA antigen-binding protein. Background Technology

[0002] Multiple myeloma (MM) is the second most common hematologic malignancy and the second leading cause of cancer death. MM is a plasma cell malignancy, often accompanied by multiple osteolytic lesions, kidney dysfunction, bone marrow infiltration, hypercalcemia, and anemia. The main current treatment for MM is systemic chemotherapy, which has severe side effects, and it is not curable.

[0003] B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor superfamily of proteins. It is mainly expressed in malignant and normal plasma cells as well as some mature B cells, but is not expressed in important tissues. It is an effective target for the treatment of MM.

[0004] With the introduction of novel drugs such as protease inhibitors (PIs), immunomodulatory drugs (IMiDs), and monoclonal antibodies (mAbs), the treatment options for multiple myeloma (MM) have expanded, but the relapse rate remains extremely high and drug resistance develops. Chimeric antigen receptor T-cell (CAR-T) therapy has shown significant efficacy and manageable toxicity for MM. However, CAR-T cell therapy targeting BCMA presents a target escape problem for MM patients with low BCMA expression. Therefore, there is a need to develop an antigen-binding protein with stronger binding activity to the BCMA antigen. Summary of the Invention

[0005] This application provides an isolated antigen-binding protein that specifically binds to BCMA protein. This application also provides a chimeric antigen receptor and fusion protein comprising the antigen-binding protein, and cells comprising and / or expressing the chimeric antigen receptor or fusion protein, wherein the cells possess one or more of the following characteristics: (1) strong amplification capacity; (2) binding activity to BCMA antigens on different cell surfaces and at different abundances; (3) high sensitivity and binding capacity for specific target cell recognition; (4) ability to kill target cells expressing BCMA; (5) secretion of cytokines upon stimulation by target cells; (6) strong proliferative capacity upon stimulation by target cells; (7) inhibition of tumor growth; and (8) good clinical therapeutic safety. The antigen-binding protein, chimeric antigen receptor, fusion protein, cells, and nucleic acid molecules provided in this application can more efficiently eliminate tumors and have therapeutic effects on both recurrent and refractory tumors, and are expected to be used in first-line, second-line, and third-line cancer treatment.

[0006] On one hand, this application provides an isolated antigen-binding protein capable of binding BCMA. The isolated antigen-binding protein comprises at least one CDR in the variable region VH of the antibody heavy chain, said VH containing the amino acid sequence shown in any one of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0007] In some embodiments, the antigen-binding protein includes an antibody or an antigen-binding fragment thereof.

[0008] In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0009] In some embodiments, the antigen-binding fragment is VHH.

[0010] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0011] In some embodiments, the antigen-binding protein includes HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:15.

[0012] In some embodiments, the antigen-binding protein includes HCDR3, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0013] In some embodiments, the antigen-binding protein includes HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:20 or SEQ ID NO:23.

[0014] In some embodiments, the antigen-binding protein includes HCDR2, and the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.

[0015] In some embodiments, the antigen-binding protein includes HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:28, SEQ ID NO:27 or SEQ ID NO:32.

[0016] In some embodiments, the antigen-binding protein includes HCDR1, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0017] In some embodiments, the antigen-binding protein includes HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0018] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0019] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0020] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0021] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0022] On the other hand, this application provides a chimeric antigen receptor comprising a targeting portion, the targeting portion including at least one antigen-binding domain targeting BCMA, and the antigen-binding domain comprising the antigen-binding protein.

[0023] In some embodiments, the chimeric antigen receptor includes a BCMA antigen-binding domain.

[0024] In some embodiments, the chimeric antigen receptor comprises two BCMA-targeting antigen-binding domains, a first antigen-binding domain and a second antigen-binding domain.

[0025] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are each independently selected from the antigen-binding protein.

[0026] On the other hand, this application provides a chimeric antigen receptor comprising a targeting portion, the targeting portion comprising at least a first targeting portion and a second targeting portion, wherein the first targeting portion targets BCMA and the second targeting portion targets GPRC5D.

[0027] In some embodiments, the first targeting portion of BCMA includes at least one antigen-binding domain targeting BCMA.

[0028] In some embodiments, the first targeting portion of BCMA includes a first antigen-binding domain targeting BCMA and a second antigen-binding domain targeting BCMA.

[0029] In some embodiments, the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA are each independently selected from the antigen-binding protein.

[0030] In some embodiments, the second targeting portion of GPRC5D includes at least one antigen-binding domain targeting GPRC5D.

[0031] In some embodiments, the second targeting portion of GPRC5D includes two antigen-binding domains targeting GPRC5D: a first antigen-binding domain targeting GPRC5D and a second antigen-binding domain targeting GPRC5D.

[0032] In some embodiments, the second targeting portion of GPRC5D includes three antigen-binding domains targeting GPRC5D: a first antigen-binding domain targeting GPRC5D, a second antigen-binding domain targeting GPRC5D, and a third antigen-binding domain targeting GPRC5D.

[0033] In some embodiments, the chimeric antigen receptor further includes a third targeting portion that targets CD38.

[0034] On the other hand, this application provides a fusion protein comprising an extracellular region and a transmembrane region, wherein the extracellular region comprises at least one antigen-binding domain targeting BCMA, and the antigen-binding domain comprises the antigen-binding protein.

[0035] In some embodiments, the extracellular region includes two BCMA-targeting antigen-binding domains, a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain are each independently selected from the antigen-binding protein.

[0036] In some embodiments, the fusion protein binds to a chimeric antigen receptor that targets GPRC5D.

[0037] In some embodiments, the chimeric antigen receptor targeting GPRC5D includes an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes at least one antigen-binding domain.

[0038] In some embodiments, the extracellular domain of the chimeric antigen receptor targeting GPRC5D has two antigen-binding domains: a first antigen-binding domain and a second antigen-binding domain.

[0039] In some embodiments, the extracellular domain of the chimeric antigen receptor targeting GPRC5D has three antigen-binding domains: a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain.

[0040] In some embodiments, the fusion protein binds to a chimeric antigen receptor that targets CD38.

[0041] In some embodiments, the chimeric antigen receptor targeting CD38 includes an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes at least one antigen-binding domain.

[0042] On the other hand, this application provides a cell that contains and / or expresses a chimeric antigen receptor or fusion protein targeting B cell maturation antigen (BCMA), a chimeric antigen receptor targeting GPRC5D, and a fusion protein targeting CD38.

[0043] In some embodiments, the chimeric antigen receptor or fusion protein targeting BCMA includes at least one antigen-binding domain targeting BCMA.

[0044] In some embodiments, the chimeric antigen receptor or fusion protein targeting BCMA includes two BCMA-targeting antigen-binding domains: a first antigen-binding domain targeting BCMA and a second antigen-binding domain targeting BCMA.

[0045] In some embodiments, the chimeric antigen receptor targeting GPRC5D includes at least one antigen-binding domain targeting GPRC5D.

[0046] In some embodiments, the antigen-binding domain targeting GPRC5D comprises two antigen-binding domains targeting GPRC5D: a first antigen-binding domain targeting GPRC5D and a second antigen-binding domain targeting GPRC5D.

[0047] In some embodiments, the antigen-binding domain targeting GPRC5D includes three antigen-binding domains targeting GPRC5D: a first antigen-binding domain targeting GPRC5D, a second antigen-binding domain targeting GPRC5D, and a third antigen-binding domain targeting GPRC5D.

[0048] In some embodiments, the CD38-targeting fusion protein includes at least one antigen-binding domain that targets CD38.

[0049] On the other hand, this application provides an isolated nucleic acid molecule encoding the antigen-binding protein, the chimeric antigen receptor or fusion protein targeting BCMA, the chimeric antigen receptor targeting GPRC5D, and / or the fusion protein targeting CD38.

[0050] In some embodiments, the nucleic acid sequence encoding a chimeric antigen receptor targeting BCMA includes a nucleic acid sequence encoding an antigen-binding domain targeting BCMA.

[0051] In some embodiments, the antigen-binding domain targeting BCMA includes at least one antigen-binding domain targeting BCMA.

[0052] In some embodiments, the antigen-binding domain targeting BCMA includes an antigen-binding domain targeting BCMA.

[0053] In some embodiments, the antigen-binding domain targeting BCMA comprises two antigen-binding domains targeting BCMA.

[0054] In some embodiments, the nucleic acid sequence encoding the chimeric antigen receptor targeting GPRC5D includes a nucleic acid sequence encoding the antigen-binding domain targeting GPRC5D.

[0055] In some embodiments, the antigen-binding domain targeting GPRC5D includes at least one antigen-binding domain targeting GPRC5D.

[0056] In some embodiments, the antigen-binding domain targeting GPRC5D includes an antigen-binding domain targeting GPRC5D.

[0057] In some embodiments, the antigen-binding domain targeting GPRC5D comprises two antigen-binding domains targeting GPRC5D.

[0058] In some embodiments, the antigen-binding domain targeting GPRC5D comprises three antigen-binding domains targeting GPRC5D.

[0059] On the other hand, this application also provides a carrier containing the aforementioned nucleic acid molecules.

[0060] On the other hand, this application provides a method for preparing modified immune cells, which includes introducing the nucleic acid molecule and / or the carrier into immune effector cells.

[0061] On the other hand, this application also provides a pharmaceutical composition comprising the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the nucleic acid molecule, the carrier, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0062] On the other hand, this application provides a method for preventing, treating and / or alleviating diseases and / or symptoms associated with BCMA, GPRC5D and / or CD38 expression, the method comprising administering to a subject in need the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition.

[0063] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0064] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0065] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0066] Figure 1 shows a schematic diagram of the BCMA-specific nanobody protein screening technology.

[0067] Figure 2 shows the polyclonal phage ELISA for panning BCMA-specific nanobody proteins.

[0068] Figure 3 shows the monoclonal phage ELISA of BCMA-specific nanobody protein panning.

[0069] Figure 4 shows the HPLC-SEC monomer purity of the BCMA-targeting nanobody.

[0070] [Correction based on Rule 91 05.11.2025] Figures 5A-5C show the SDS-PAGE monomer purity of the BCMA-targeting nanobody.

[0071] Figure 6 shows the binding activity of the BCMA-targeting nanobody to the BCMA protein.

[0072] Figure 7 shows the expression level of BCMA antigen in different MM target cell lines as detected by quantitative flow cytometry.

[0073] Figure 8 shows the flow cytometry binding activity of the BCMA-targeting nanobody to CHO-hBCMA cells.

[0074] Figure 9 shows the flow cytometry binding activity of the BCMA-targeting nanobody to NCIH929 cells.

[0075] Figure 10 shows the flow cytometry binding activity of the BCMA-targeting nanobody to MOLP8 cells.

[0076] Figure 11 shows the SPR affinity level of the BCMA-targeting nanobody.

[0077] Figure 12 shows the crystal structure of the extracellular domain ECD of human BCMA.

[0078] Figure 13 shows the binding activity of the BCMA-targeting nanobody with the linear epitope peptide.

[0079] Figure 14 shows the identification of SPR epitope differences between the BCMA-targeting nanobody and NanoBCMA9.2.

[0080] Figure 15 shows the identification of SPR epitope differences between exemplary NanoBCMA9.2 and hBCMA22.

[0081] Figure 16 shows the biotinylation verification and binding activity of an exemplary BCMA-targeting nanobody.

[0082] [Correction based on Rule 91 05.11.2025] Figures 17A-17B show the identification of FCM epitope differences in exemplary BCMA-targeting nanobodies.

[0083] Figure 18 shows the identification of FCM epitope differences between the exemplary BCMA-targeting nanobody and NanoBCMA9.2.

[0084] Figure 19 shows the polymerization potential of an exemplary BCMA-targeting nanobody induced by BCMAECD protein.

[0085] [Correction based on Rule 91 05.11.2025] Figure 20A shows the non-specific binding activity of an exemplary BCMA-targeting nanobody with CHO cells (which do not express BCMA); Figure 20B shows the non-specific binding activity of an exemplary BCMA-targeting nanobody with different non-specific target cells.

[0086] Figure 21 shows a schematic diagram of the structural connections of an exemplary BCMA-specific chimeric antigen receptor.

[0087] Figure 22 shows a schematic diagram of the third-generation lentivirus vector system.

[0088] Figure 23 shows an exemplary BCMACAR lentiviral transduction of Day 5 T cell surface CAR expression.

[0089] Figure 24 shows the positivity rate at different days after transducing T cells with the exemplary BCMACAR.

[0090] Figure 25 shows the cell viability at different days after transducing exemplary BCMACAR T cells.

[0091] Figure 26 shows the cell volume at different days after exemplary BCMACAR transduction of T cells.

[0092] Figure 27 shows the in vitro proliferation curve of an exemplary BCMACAR-T cell.

[0093] Figure 28 shows the killing efficiency of exemplary BCMACAR-T cells at different effector-to-target ratios.

[0094] Figure 29 shows the IL-2 secretion level of exemplary BCMACAR-T cells co-incubated with target cells.

[0095] Figure 30 shows the IFN-γ secretion level of exemplary BCMACAR-T cells after co-incubation with target cells.

[0096] Figure 31 shows the proportion of residual tumor cells in exemplary BCMACAR-T cells after co-incubation with MOLP8 target cells.

[0097] Figure 32 shows the CAR positivity rate of exemplary BCMACAR-T cells under co-incubation with MOLP8 target cells.

[0098] Figure 33 shows the statistical results of CAR positivity rate of exemplary BCMACAR-T cells under co-incubation with MOLP8 target cells.

[0099] Figure 34 shows an exemplary flow cytometry analysis of CD4 / CD8 subtypes in BCMACAR-T cells.

[0100] Figure 35 shows the CD4 / CD8 subtype statistics of exemplary BCMACAR-T cells.

[0101] Figure 36 shows an exemplary flow cytometry analysis of BCMACAR-T cell memory phenotypes.

[0102] Figure 37 shows the statistics of exemplary BCMACAR-T cell memory phenotypes.

[0103] Figure 38 shows the expression level of activating receptors in exemplary BCMACAR-T cells.

[0104] Figure 39 shows the exhaustion receptor expression level in exemplary BCMACAR-T cells.

[0105] Figure 40 shows an exemplary detection of CAR expression on the surface of T cells transduced by anti-BCMA bivalent CAR lentivirus.

[0106] Figure 41 shows the in vitro proliferation curve of an exemplary anti-BCMA bivalent CAR-T cell.

[0107] Figure 42 shows the cell viability at different days after exemplary anti-BCMA bivalent CAR transduction of T cells.

[0108] Figures 43-44 show the killing efficiency of exemplary anti-BCMA bivalent CAR-T cells with different effector-to-target ratios in the presence or absence of sBCMA.

[0109] Figure 45 shows the secretion of IFN-γ by exemplary anti-BCMA bivalent CAR-T cells after co-incubation with target cells in the presence or absence of sBCMA.

[0110] Figures 46-48 show the functional assay of exemplary anti-BCMA bivalent CAR-T cells under repeated stimulation by target cells.

[0111] Figure 49 shows the tumor suppression effect in NSG mice using the orthotopic transplantation model.

[0112] Figure 50 shows the trend of body weight change in the orthotopic transplantation model NSG mice.

[0113] Figures 51-58 show schematic diagrams of the structural connections of an exemplary multispecific chimeric antigen receptor.

[0114] Figure 59 shows the antigen expression levels of different MM target cell lines predicted by DepMap Data Explorer.

[0115] Figure 60 shows the antigen expression levels of different MM target cell lines detected by flow cytometry.

[0116] Figure 61 shows an exemplary flow cytometry assay for multispecific chimeric antigen receptor T-cell lentivirus titers.

[0117] Figure 62 shows the in vitro proliferation curves of exemplary multispecific chimeric antigen receptor transduced T cells at different days.

[0118] Figure 63 shows the cell viability at different days after exemplary multispecific chimeric antigen receptor-guided T cells.

[0119] Figures 64-69 show the killing efficiency of exemplary multispecific CAR-T cells at different effector-to-target ratios compared to target cells in the presence of sBCMA.

[0120] Figure 70 shows the killing activity of exemplary multispecific CAR-T cells against CD38 single-positive target cells.

[0121] Figure 71 shows the cytokine secretion levels of exemplary multispecific CAR-T cells stimulated by NCIH929 target cells.

[0122] Figure 72 shows an exemplary multispecific CAR-T cell assay in NCIH929-BCMA. KO Cytokine secretion levels under target cell stimulation.

[0123] Figure 73 shows the cytokine secretion levels of exemplary multispecific CAR-T cells stimulated by MOLP8 target cells.

[0124] Figure 74 shows the cytotoxic activity of exemplary multispecific CAR-T cells against target cells in a long-term assay.

[0125] Figure 75 shows the positive rate of CAR molecule expression in exemplary multispecific CAR-T cells stimulated by target cells in a long-term assay.

[0126] Figure 76 shows the CAR molecule expression level of exemplary multispecific CAR-T cells under target cell stimulation in a long-term assay.

[0127] Figure 77 shows the target proliferation fold of exemplary multispecific CAR-T cells stimulated by target cells in a long-term assay.

[0128] Figure 78 shows the IFN-γ secretion level of exemplary multispecific CAR-T cells under target cell stimulation in a long-term assay.

[0129] Figure 79 shows the IL-2 secretion level of exemplary multispecific CAR-T cells under target cell stimulation in a long-term assay.

[0130] Figure 80 shows the IL-6 secretion level induced by target cell stimulation in an exemplary multispecific CAR-T cell co-incubation system with monocytes. Here, T represents CAR-T cells cultured alone, E represents target cells, and M represents CD14+ monocytes.

[0131] Figure 81 shows an exemplary statistical representation of the memory phenotype of multispecific CAR-T cells.

[0132] Figure 82 shows a schematic diagram of the structural connections of an exemplary bispecific chimeric antigen receptor.

[0133] Figure 83 shows the expression of CAR on the surface of different T cells during Day 5 of exemplary bispecific CAR lentiviral transduction.

[0134] Figures 84-86 show the killing efficiency of exemplary bispecific CAR-T cells under different target cells and different effector-to-target ratios.

[0135] Figure 87 shows the verification of mutual interference between antigen recognition domains of exemplary bispecific CAR-T cells in different target cells.

[0136] [Corrected according to Rule 91 05.11.2025] Figure 88A shows the cytokine secretion of exemplary bispecific CAR-T cells after co-incubation with early line MM cells NCIH929 in the absence of sBCMA; Figure 88B shows the cytokine secretion of exemplary bispecific CAR-T cells after co-incubation with midline MM cells MM1S in the absence of sBCMA; Figure 88C shows the cytokine secretion of exemplary bispecific CAR-T cells after co-incubation with late line MM cells MOLP8 in the absence of sBCMA. [0136.1] [Correction 05.11.2025 according to Rule 91] Figure 89A shows the cytokine secretion of exemplary bispecific CAR-T cells after co-incubation with early line MM cells NCIH929 under sBCMA conditions; Figure 89B shows the cytokine secretion of exemplary bispecific CAR-T cells after co-incubation with midline MM cells MM1S under sBCMA conditions; Figure 89C shows the cytokine secretion of exemplary bispecific CAR-T cells after co-incubation with late line MM cells MOLP8 under sBCMA conditions.

[0137] Figure 90 shows the cytotoxic activity of exemplary bispecific CAR-T cells against target cells and the positive rate of CAR molecule expression in a long-term assay.

[0138] Figure 91 shows the consistency test between the actual configured pH value and the theoretical set value.

[0139] Figures 92-94 show the validation results of exemplary bispecific CAR-T cells under reducing conditions and low pH interference. Detailed Implementation

[0140] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0141] Terminology Definition

[0142] In this application, the term "isolated antigen-binding protein" generally refers to a protein that has antigen-binding ability after being removed from its native state. The "isolated antigen-binding protein" may include an antigen-binding portion and optionally, a framework or scaffold portion that allows the antigen-binding portion to adopt a conformation that promotes its antigen-binding. The antigen-binding protein may include, for example, an antibody-derived protein framework region (FR) or an alternative protein framework region or artificial framework region having a transplanted variable region (CDR) or a CDR derivative. For example, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof. For example, the antigen-binding protein may bind to the GPRC5D protein. For example, the antigen-binding protein may compete with a reference antibody for binding to the GPRC5D protein. For example, the antigen-binding protein may include an antibody heavy chain variable region (VH). For example, the antigen-binding protein may include at least one CDR derived from the antibody heavy chain variable region (VH). For example, the VH may include HCDR3, HCDR2, and / or HCDR1. For example, the antigen-binding protein may be a VHH. For example, the antigen-binding protein may include an antibody heavy chain constant region, which may be derived from IgG. For example, the antibody heavy chain constant region may be derived from human IgG. For example, the antibody heavy chain constant region may be derived from human IgG1.

[0143] In this application, the term "variable" generally refers to the fact that certain segments of the variable domain may differ significantly in sequence between antibodies. The variable domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire variable domain. It is typically concentrated in three segments within the light and heavy chain variable domains, known as hypervariable regions (CDRs or HVRs). The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, most of which are β-sheet conformations linked by three CDRs forming a ring link, and in some cases forming part of a β-sheet structure. The CDRs in each chain are held together closely by the FR regions, and CDRs from the other chain together promote the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

[0144] The term "antibody" as used includes both the complete antibody and its binding fragment. Typically, the fragment competitively binds to the antigen specifically, as derived from the complete antibody. Optionally, the antibody or its binding fragment may chemically bind to other proteins or be expressed as a fusion protein with other proteins. For example, the antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody. For example, the binding protein of the antibody or its binding fragment may include BCMA. For example, the antibody or its binding fragment may be specific to BCMA.

[0145] The term "antigen-binding fragment" refers to a portion of a complete antibody and specifically to the antigen-determining variable region of the complete antibody. For example, the antigen-binding fragment may include Fab, Fab', F(ab')2, Fv fragments and single-chain Fv fragments, tandem Fv fragments, VHH, and bispecific antibodies. For example, the antigen-binding fragment may be a VHH. For example, the antigen-binding fragment may bind to BCMA. For example, the antigen-binding fragment may be specific to BCMA.

[0146] In this application, the term "VHH" generally refers to an antibody containing a variable antigen-binding domain of a heavy chain antibody. VHH may also be referred to as a nanobody (Nb) and / or a single-domain antibody. For example, the VHH may bind to BCMA. For example, the VHH may be specific for BCMA.

[0147] In this application, the term "chimeric antigen receptor" (CAR) generally refers to a recombinant polypeptide comprising at least an extracellular domain, a transmembrane region, and an intracellular domain that specifically bind to an antigen or target. For example, a hinge region may be included between the extracellular domain and the transmembrane region. For example, the chimeric antigen receptor may also include a low-density lipoprotein receptor-associated protein or a fragment thereof. For example, the chimeric antigen receptor may include a signal peptide. The binding of the extracellular domain of the CAR to the target antigen on the surface of the target cell leads to CAR clustering and delivers activation stimuli to CAR-containing cells. The CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis, and / or production of molecules capable of mediating cell death expressing the target antigen in a major histocompatibility (MHC)-independent manner. For example, the extracellular structure may include the antigen-binding protein described above. For example, the extracellular structure may specifically bind BCMA.

[0148] In this application, the terms "intracellular domain" and "intracellular region" have the same meaning, both referring to intracellular domains that include any truncated portion sufficient to transduce activation signals. The intracellular domain may include intracellular signaling regions and / or co-stimulatory signaling regions. The term "intracellular signaling region" refers to an intracellular region capable of generating signals that promote the function of immune effectors in CAR-containing cells (e.g., CAR-T cells or NK cells expressing CAR). For example, the intracellular signaling region may include intracellular signaling regions of one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and domains containing at least one ITAM. For example, the intracellular signaling region may be a signal transduction domain derived from CD3ζ. The term "co-stimulatory signaling region" refers to a portion of the intracellular signaling region that is capable of transducing effector signals. For example, the co-stimulatory signaling region may include intracellular co-stimulatory signaling regions derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, ligands of CD83, CD40, and MyD88. For example, the co-stimulatory signaling region may be an intracellular co-stimulatory signaling region derived from 4-1BB. For example, the co-stimulatory signaling region may be an intracellular co-stimulatory signaling region derived from CD28.

[0149] In this application, the terms "transmembrane region" and "transmembrane domain" have the same meaning, both referring to structural domains of peptides, polypeptides, or proteins that can cross the cell membrane. These domains can be used to anchor extracellular structural domains to the cell membrane. For example, the transmembrane region may comprise a transmembrane domain of one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region may be derived from the transmembrane region of CD8. For example, the transmembrane region may be derived from the transmembrane region of CD28.

[0150] In this application, the term "hinge region" refers to a portion of an antibody heavy chain polypeptide that connects the CH1 and CH2 domains, for example, from approximately position 216 to approximately position 230 according to the Kabat EU numbering system. A hinge region is typically a dimer molecule composed of two polypeptides having the same amino acid sequence. A hinge region generally comprises approximately 25 amino acid residues and is flexible, allowing independent movement of the antigen-binding region. A hinge region can be subdivided into three domains: upper, middle, and lower hinge domains. For example, the hinge region may comprise a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge area may be derived from the hinge area of ​​CD8.

[0151] In this application, the term "low-density lipoprotein receptor-related protein" refers to a cell surface protein belonging to the endocytic receptor family. It is widely distributed in organisms and exhibits significant interstitial variability. Its main function is to take up cholesterol into cells for cell proliferation and the synthesis of steroid hormones and bile salts. For example, the low-density lipoprotein receptor-related protein can be derived from any vertebrate. For example, the low-density lipoprotein receptor-related protein or a fragment thereof can be located at the C-terminus of the intracellular signaling region. For example, the low-density lipoprotein receptor-related protein or a fragment thereof can comprise one or more selected from the group consisting of low-density lipoprotein receptor-related proteins 1-12 and their functional fragments. For example, the low-density lipoprotein receptor-related protein or a fragment thereof can be low-density lipoprotein receptor-related protein 6 or a fragment thereof.

[0152] In this application, the term "signal peptide" refers to a leader sequence located at the N-terminus of a nascent CAR protein that guides the nascent protein to the endoplasmic reticulum for subsequent surface expression during or after translation. For example, the signal peptide may be derived from the signal peptide of the CD8 protein.

[0153] In this application, the term "fusion protein" generally refers to a protein composed of two or more polypeptides that, although not normally bound in their native state, are linked together by peptide bonds through their respective amino and carboxyl terminals to form a continuous polypeptide. It should be understood that the two or more polypeptide components can bind directly or indirectly through peptide linkers / spacers. In this application, the fusion protein includes extracellular domains and transmembrane regions that specifically bind antigens or targets. For example, the fusion protein can specifically bind BCMA. For example, the fusion protein can specifically bind CD38. In this application, the fusion protein may also include a co-stimulatory signaling region. In this application, the fusion protein can bind to a chimeric antigen receptor.

[0154] In this application, the term "isolated nucleic acid molecule" includes DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. The term "promoter" generally refers to a DNA sequence that can regulate the expression of a selected DNA sequence operatively linked to a promoter, thereby affecting the expression of the selected DNA sequence in a cell. For example, the nucleic acid molecule may encode a protein including its host antigen-binding protein and / or the chimeric antigen receptor. For example, the nucleic acid molecule may include a promoter. For example, the promoter may be a constitutive promoter. For example, the promoter may be the EF1α promoter.

[0155] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle that can insert a polynucleotide encoding a protein into itself and enable the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material elements they carry to be expressed within the host cells. For example, vectors can include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors can include retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. The carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0156] In this application, the term "directly linked" is used in contrast to the term "indirectly linked." "Directly linked" generally refers to a direct connection between substances. For example, a direct connection can be a situation where substances are directly linked without a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirectly linked" generally refers to a situation where substances are not directly linked. For example, an indirect connection can be a connection via a spacer.

[0157] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this invention. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells may include cells transfected in vitro using the vectors described in this application. Cells may be bacterial cells (e.g., *Escherichia coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or stem cells (e.g., ES cells, iPS cells, mesenchymal stem cells) or immune cells (e.g., T cells, NK cells, NKT cells, macrophages).

[0158] In this application, the term "pharmaceutical composition" generally refers to a chemical or biological composition suitable for administration to a mammalian individual. For example, the pharmaceutical composition may include the antigen-binding protein, the chimeric antigen receptor, the polypeptide, the nucleic acid molecule, the carrier and / or the cell, and optionally a pharmaceutically acceptable carrier. The pharmaceutical composition may be used to prevent, treat, and / or alleviate diseases or conditions associated with BCMA expression. For example, diseases or conditions associated with BCMA expression may include tumors. For example, the tumor may also express GPRC5D. For example, the tumor may also express CD38. For example, the tumor includes solid tumors and / or non-solid tumors. For example, the tumor may include hematologic malignancies and / or lymphomas. For example, the tumor may include myeloma.

[0159] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the doses and concentrations employed. Physiologically acceptable carriers may include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) peptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming anti-charge ions such as sodium; and / or nonionic surfactants.

[0160] In this application, the term "tumor" generally refers to a growth or solid lesion formed by abnormal cell growth. In this application, a tumor can be a solid tumor or a hematologic malignancy. For example, in this application, a tumor can be a tumor expressing BCMA. For example, a tumor can be a tumor expressing BCMA and GPRC5D. For example, a tumor can be a tumor expressing BCMA, GPRC5D, and CD38.

[0161] In this application, the proteins, peptides and / or amino acid sequences involved should also be understood to include at least the following range: variants or homologs that have the same or similar functions as the said protein or peptide.

[0162] In this application, the variant can be, for example, a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to BCMA protein or a fragment thereof). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or polypeptide prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conserved substitution.

[0163] In this application, the homolog can be a protein or polypeptide having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody that specifically binds to BCMA protein or a fragment thereof).

[0164] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated by comparing two sequences to be aligned within a comparison window, determining the number of positions in the two sequences containing the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence homology percentage. Alignments performed to determine the sequence homology percentage can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. Homology can also be determined using FASTA and BLAST. A description of the FASTA algorithm can be found in W.R. Pearson and DJ Lipman, “An Improved Tool for Biological Sequence Alignment,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and DJ Lipman and W.R. Pearson, “A Fast and Sensitive Search for Protein Similarity,” Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E.W. Myers, and D. Lipman, “A Basic Tool for Local Alignment Search,” Journal of Molecular Biology, 215: 403-410, 1990.

[0165] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0166] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0167] Invention Details

[0168] Isolated antigen-binding proteins

[0169] On the one hand, this application provides an isolated antigen-binding protein capable of binding to B cell maturation antigen (BCMA). For example, the isolated antigen-binding protein can specifically bind to human BCMA protein expressed on the cell surface.

[0170] In this application, the antigen-binding protein may include an antibody or an antigen-binding fragment thereof. In this application, the antigen-binding fragment may be Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb. In this application, the antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

[0171] CDR

[0172] The CDR (Complementarity Determinant Region) of an antibody, also known as the complementarity-determining region, is part of the variable region. Amino acid residues in this region can contact antigens or antigenic epitopes. Antibody CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and a combination of Kabat / Chothia. These coding systems are known in the art and can be found, for example, at http: / / www.bioinf.org.uk / abs / index.html#kabatnum. Those skilled in the art can determine the CDR region using different coding systems based on the antibody's sequence and structure. The CDR region may differ when using different coding systems. In this application, the term CDR encompasses CDR sequences partitioned according to any CDR partitioning method; it also encompasses variants of the CDR, including amino acid sequences with substitutions, deletions, and / or additions of one or more amino acids. For example, substitutions, deletions, and / or insertions of 1-30, 1-20, or 1-10 amino acids, or 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids; this also covers homologs, which can be amino acid sequences having at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or higher) sequence homology to the amino acid sequence of the CDR. In this application, the isolated antigen-binding protein is defined using the IMGT coding system.

[0173] In this application, the isolated antigen-binding protein comprises at least one CDR in the variable region VH of the antibody heavy chain, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0174] In this application, the antigen-binding protein may comprise HCDR3, which comprises the amino acid sequence shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:15. In some embodiments, the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.

[0175] In this application, the antigen-binding protein may include HCDR2, and HCDR2 comprises the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:20, or SEQ ID NO:23. In some embodiments, HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23.

[0176] In this application, the antigen-binding protein may include HCDR1, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:28, SEQ ID NO:27, or SEQ ID NO:32. In one embodiment, HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0177] In this application, the antigen-binding protein may include HCDR1, HCDR2, and HCDR3, and the antigen-binding protein comprises any one of the following amino acid sequences:

[0178] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0179] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0180] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0181] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0182] In some embodiments, the antigen-binding protein comprises any one of the following amino acid sequences:

[0183] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0184] (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2;

[0185] (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2;

[0186] (4) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:21, and HCDR3: SEQ ID NO:3;

[0187] (5) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:21, and HCDR3: SEQ ID NO:4;

[0188] (6) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:5;

[0189] (7) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:6

[0190] (8) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:7;

[0191] (9) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:8;

[0192] (10) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:9;

[0193] (11) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:10;

[0194] (12) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:11;

[0195] (13) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:12;

[0196] (14) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:13;

[0197] (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14;

[0198] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0199] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0200] FR

[0201] In this application, the antibody framework region FR refers to the portion of the antibody variable region located between more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frames 1 to 4 (FR1, FR2, FR3, and FR4) and provide a backbone for presenting six CDRs (three from the heavy chain and three from the light chain) in three-dimensional space to form an antigen-binding surface.

[0202] In this application, the isolated antigen-binding protein may include H-FR1, the C-terminus of which is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 contains the amino acid sequence shown in SEQ ID NO:33.

[0203] In this application, the antigen-binding protein may include H-FR2, which is located between HCDR1 and HCDR2, and H-FR2 may contain the amino acid sequence shown in SEQ ID NO:34 or SEQ ID NO:37.

[0204] In this application, the antigen-binding protein may include H-FR3, which is located between HCDR2 and HCDR3, and the H-FR3 may contain the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:38.

[0205] In this application, the antigen-binding protein may include H-FR4, the N-terminus of which is directly or indirectly linked to the C-terminus of HCDR3, and the H-FR4 contains the amino acid sequence shown in SEQ ID NO:36.

[0206] In this application, the antigen-binding protein may comprise H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1 comprises the amino acid sequence shown in SEQ ID NO:33, H-FR2 comprises the amino acid sequence shown in SEQ ID NO:34 or SEQ ID NO:37, H-FR3 comprises the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:38, and H-FR4 comprises the amino acid sequence shown in SEQ ID NO:36.

[0207] For example, the antigen-binding protein comprises H-FR1, H-FR2, H-FR3, and H-FR4, wherein H-FR1, H-FR2, H-FR3, and H-FR4 are selected from the amino acid sequences shown in any one of the following groups:

[0208] (1) H-FR1 contains the amino acid sequence shown in SEQ ID NO:33, H-FR2 contains the amino acid sequence shown in SEQ ID NO:34, H-FR3 contains the amino acid sequence shown in SEQ ID NO:35, and H-FR4 contains the amino acid sequence shown in SEQ ID NO:36;

[0209] (2) H-FR1 contains the amino acid sequence shown in SEQ ID NO:33, H-FR2 contains the amino acid sequence shown in SEQ ID NO:37, H-FR3 contains the amino acid sequence shown in SEQ ID NO:35, and H-FR4 contains the amino acid sequence shown in SEQ ID NO:36; and

[0210] (3) H-FR1 contains the amino acid sequence shown in SEQ ID NO:33, H-FR2 contains the amino acid sequence shown in SEQ ID NO:37, H-FR3 contains the amino acid sequence shown in SEQ ID NO:38, and H-FR4 contains the amino acid sequence shown in SEQ ID NO:36.

[0211] VH / VHH

[0212] In this application, the isolated antigen-binding protein may include a heavy chain variable region VH, and the VH may be an amino acid sequence shown in any one of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0213] In this application, the isolated antigen-binding protein may contain an antibody or an antigen-binding fragment thereof.

[0214] For example, the antigen-binding fragments include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

[0215] For example, the antigen-binding fragment is camel-derived, chimeric, partially humanized, and / or fully humanized.

[0216] In this application, the antigen-binding fragment may be VHH, wherein the VHH comprises the amino acid sequence shown in any one of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0217] Heavy chain constant region

[0218] In this application, the isolated antigen-binding protein may include a heavy chain constant region. The heavy chain constant region refers to a region containing at least three heavy chain constant domains CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy chain constant region corresponds to an antibody isotype. For example, an antibody containing a γ constant region is an IgG antibody, an antibody containing a δ constant region is an IgD antibody, and an antibody containing an α constant region is an IgA antibody. Furthermore, an antibody containing a μ constant region is an IgM antibody, and an antibody containing an ε constant region is an IgE antibody. Certain isotypes may be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgG1 (containing a γ1 constant region), IgG2 (containing a γ2 constant region), IgG3 (containing a γ3 constant region), and IgG4 (containing a γ4 constant region) antibodies; IgA antibodies include, but are not limited to, IgA1 (containing an α1 constant region) and IgA2 (containing an α2 constant region) antibodies; IgM includes, but is not limited to, IgM1 and IgM2.

[0219] In this application, the antigen-binding protein may include an antibody heavy chain constant region, which may be derived from IgG. In this application, the antigen-binding protein may include an antibody heavy chain constant region, which may be derived from human IgG. In this application, the antigen-binding protein may include an antibody heavy chain constant region, which may be derived from human IgG1.

[0220] Chimeric antigen receptor

[0221] On the other hand, this application provides a chimeric antigen receptor (CAR) comprising a targeting portion, said targeting portion including at least one antigen-binding domain targeting BCMA. For example, the antigen-binding domain targeting BCMA may comprise the antigen-binding protein described in this application.

[0222] In some embodiments, it includes a BCMA-targeting antigen-binding domain comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0223] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0224] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0225] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0226] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0227] In some embodiments, it includes a BCMA-targeting antigen-binding domain comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0228] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0229] (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2;

[0230] (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2;

[0231] (4) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 3;

[0232] (5) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 4;

[0233] (6) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 5;

[0234] (7) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 6

[0235] (8) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 7;

[0236] (9) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 8;

[0237] (10) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 9;

[0238] (11) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 10;

[0239] (12) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 11;

[0240] (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 12;

[0241] (14) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 13;

[0242] (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14;

[0243] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0244] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0245] In some embodiments, it includes two BCMA-targeting antigen-binding domains, a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain are each independently selected from the antigen-binding protein described in this application.

[0246] In one embodiment, the first antigen-binding domain and the second antigen-binding domain target the same epitope of BCMA. In one embodiment, the first antigen-binding domain and the second antigen-binding domain target different epitopes of BCMA. In one embodiment, the first antigen-binding domain and the second antigen-binding domain comprise the same or different amino acid sequences.

[0247] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are directly or indirectly linked. In one embodiment, the first antigen-binding domain and the second antigen-binding domain are linked by a linker. For example, the linker is a linker peptide. For example, the linker peptide contains the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linker peptide is GGGGS (SEQ ID NO: 113). For example, the linker peptide is (GGGGS)3 (SEQ ID NO: 114). For example, the linker peptide contains the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0248] In some embodiments, the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain comprise amino acid sequences selected from the group consisting of:

[0249] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16;

[0250] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:17.

[0251] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18.

[0252] (4) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0253] (5) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:17, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16.

[0254] (6) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17;

[0255] (7) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:27, HCDR2 is SEQ ID NO:20, and HCDR3 is SEQ ID NO:18;

[0256] (8) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:32, HCDR2 is SEQ ID NO:23, and HCDR3 is SEQ ID NO:15;

[0257] (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16.

[0258] (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:17.

[0259] (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18;

[0260] (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0261] (13) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16.

[0262] (14) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:17.

[0263] (15) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18; and

[0264] (16) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15.

[0265] In some embodiments, the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain comprise amino acid sequences selected from the group consisting of:

[0266] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0267] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0268] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0269] (4) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0270] (5) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0271] (6) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0272] (7) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0273] (8) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0274] (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0275] (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0276] (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0277] (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0278] (13) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0279] (14) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0280] (15) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0281] (16) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0282] (17) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0283] (18) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0284] (19) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0285] (20) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2.

[0286] (21) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2.

[0287] (22) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0288] (23) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0289] (24) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0290] (25) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0291] (26) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0292] (27) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0293] (28) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0294] (29) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13.

[0295] (30) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0296] (31) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0297] (32) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0298] (33) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2.

[0299] (34) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0300] (35) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; and

[0301] (36) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15.

[0302] In some embodiments, the CAR may include an intracellular co-stimulatory domain that can provide a stimulatory signal. For example, the co-stimulatory domain may include an intracellular co-stimulatory domain of one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. For example, the co-stimulatory signaling region is an intracellular co-stimulatory signaling region derived from 4-1BB or CD28. For example, the amino acid sequence of the co-stimulatory signaling region is shown in SEQ ID NO:120 or SEQ ID NO:121.

[0303] In this application, the CAR may include an intracellular signaling region, which can transmit activation signals into the cell. For example, the intracellular signaling region may include an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and other domains containing at least one ITAM. For example, the intracellular signaling region is a signaling domain derived from CD3ζ. For example, the amino acid sequence of the intracellular signaling region is shown in SEQ ID NO:122.

[0304] In this application, the CAR may include a transmembrane domain, which is a sequence of a cell surface protein that crosses the cell membrane and may contain a hydrophobic alpha helix. The transmembrane domain may originate from any type I transmembrane protein. The transmembrane domain may be a synthetic sequence predicted to form a hydrophobic helix. For example, the transmembrane region may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, DAP10, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region may be a transmembrane region derived from CD8 or CD28. For example, the amino acid sequence of the transmembrane region is shown in SEQ ID NO:118 or SEQ ID NO:119.

[0305] In this application, the CAR may include a hinge region located between the extracellular targeting portion and the transmembrane domain. For example, the hinge region may comprise a hinge region of one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. For example, the hinge region may be derived from CD8 or CD28. For example, the amino acid sequence of the hinge region may be as shown in SEQ ID NO:116 or SEQ ID NO:117.

[0306] In some embodiments, the CAR further comprises low-density lipoprotein receptor-associated protein or a fragment thereof, the low-density lipoprotein receptor-associated protein or a fragment thereof located at the C-terminus of the intracellular signaling region.

[0307] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof may comprise one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments.

[0308] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof may be low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

[0309] For example, the low-density lipoprotein receptor-associated protein or a fragment thereof is the Ori2 protein (self-constructed by Shanghai Yuaneng Cell Medicine Technology Co., Ltd.). For example, the amino acid sequence of the Ori2 protein is shown in SEQ ID NO:124.

[0310] In this application, the CAR may comprise a signal peptide. For example, the signal peptide may be a signal peptide derived from the CD8 protein. For example, the amino acid sequence of the signal peptide is shown in SEQ ID NO:115.

[0311] In this application, the CAR from the N-terminus to the C-terminus can be: a CD8 protein signal peptide, an extracellular antigen-binding domain, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular co-stimulatory signaling region, a CD3ζ signal transduction domain, and an Ori2 protein.

[0312] In some embodiments, the extracellular antigen-binding domain may include an antigen-binding domain that targets BCMA.

[0313] For example, the extracellular antigen binding domain may be SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, or SEQ ID NO:72.

[0314] In some embodiments, the extracellular antigen-binding domain may include a first antigen-binding domain targeting BCMA, a linker, and a second antigen-binding domain targeting BCMA.

[0315] In some embodiments, the first antigen-binding domain and the second antigen-binding domain may each be independently selected from the group consisting of: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72. In some embodiments, the linker may be an amino acid sequence of (GGGGS)n.

[0316] For example, the extracellular antigen-binding domain can be composed of the N-terminus to the C-terminus as follows:

[0317] (1) The first antigen-binding domain is SEQ ID NO:40, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:44.

[0318] (2) The first antigen-binding domain is SEQ ID NO:40, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:44.

[0319] (3) The first antigen-binding domain is SEQ ID NO:40, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:62.

[0320] (4) The first antigen-binding domain is SEQ ID NO:40, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:62.

[0321] (5) The first antigen-binding domain is SEQ ID NO:44, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:40.

[0322] (6) The first antigen-binding domain is SEQ ID NO:44, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:40.

[0323] (7) The first antigen-binding domain is SEQ ID NO:44, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:70.

[0324] (8) The first antigen-binding domain is SEQ ID NO:44, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:70.

[0325] (9) The first antigen-binding domain is SEQ ID NO:44, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:42.

[0326] (10) The first antigen-binding domain is SEQ ID NO:44, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:42.

[0327] (11) The first antigen-binding domain is SEQ ID NO:62, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:40.

[0328] (12) The first antigen-binding domain is SEQ ID NO:62, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:40.

[0329] (13) The first antigen-binding domain is SEQ ID NO:62, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:70.

[0330] (14) The first antigen-binding domain is SEQ ID NO:62, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:70.

[0331] (15) The first antigen-binding domain is SEQ ID NO:70, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:44.

[0332] (16) The first antigen-binding domain is SEQ ID NO:70, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:44.

[0333] (17) The first antigen-binding domain is SEQ ID NO:70, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:42.

[0334] (18) The first antigen-binding domain is SEQ ID NO:70, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:42.

[0335] (19) The first antigen-binding domain is SEQ ID NO:70, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:66.

[0336] (20) The first antigen-binding domain is SEQ ID NO:70, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:66.

[0337] (21) The first antigen-binding domain is SEQ ID NO:42, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:44.

[0338] (22) The first antigen-binding domain is SEQ ID NO:42, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:44.

[0339] (23) The first antigen-binding domain is SEQ ID NO:42, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:70.

[0340] (24) The first antigen-binding domain is SEQ ID NO:42, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:70.

[0341] (25) The first antigen-binding domain is SEQ ID NO:42, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:66.

[0342] (26) The first antigen-binding domain is SEQ ID NO:42, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:66.

[0343] (27) The first antigen-binding domain is SEQ ID NO:66, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:70.

[0344] (28) The first antigen-binding domain is SEQ ID NO:66, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:70.

[0345] (29) The first antigen-binding domain is SEQ ID NO:66, the linker is GGGGS, and the second antigen-binding domain is SEQ ID NO:42, or

[0346] (30) The first antigen-binding domain is SEQ ID NO:66, the linker is (GGGGS)3, and the second antigen-binding domain is SEQ ID NO:42.

[0347] In this application, the targeting portion of the chimeric antigen receptor may include a first targeting portion and a second targeting portion, wherein the first targeting portion targets BCMA and the second targeting portion targets GPRC5D.

[0348] In some embodiments, the first targeting portion of BCMA includes at least one antigen-binding domain targeting BCMA. In some embodiments, the first targeting portion of BCMA may comprise the antigen-binding protein described in this application.

[0349] In some embodiments, the first targeting portion of BCMA includes an antigen-binding domain targeting BCMA. In some embodiments, the first targeting portion of BCMA includes HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0350] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0351] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0352] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0353] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0354] For example, the antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0355] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0356] (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2;

[0357] (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2;

[0358] (4) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 3;

[0359] (5) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 4;

[0360] (6) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 5;

[0361] (7) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 6

[0362] (8) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 7;

[0363] (9) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 8;

[0364] (10) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 9;

[0365] (11) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 10;

[0366] (12) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 11;

[0367] (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 12;

[0368] (14) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 13;

[0369] (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14;

[0370] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0371] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0372] For example, the first targeting portion targeting BCMA may be SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, or SEQ ID NO:72.

[0373] In some embodiments, the first targeting portion of BCMA includes a first antigen-binding domain targeting BCMA and a second antigen-binding domain targeting BCMA.

[0374] In some embodiments, the first antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and the first and second antigen-binding domains targeting BCMA are each independently selected from any one of the following amino acid sequences:

[0375] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0376] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0377] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0378] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0379] For example, the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA are each independently selected from any one of the following groups of amino acid sequences:

[0380] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0381] (2) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:2;

[0382] (3) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:21, and HCDR3: SEQ ID NO:2;

[0383] (4) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:21, and HCDR3: SEQ ID NO:3;

[0384] (5) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:21, and HCDR3: SEQ ID NO:4;

[0385] (6) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:5;

[0386] (7) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:6

[0387] (8) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:7;

[0388] (9) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:8;

[0389] (10) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:9;

[0390] (11) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:10;

[0391] (12) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:11;

[0392] (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 12;

[0393] (14) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 13;

[0394] (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14;

[0395] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0396] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0397] For example, the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA can each be independently selected from the group consisting of: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0398] In some embodiments, the second targeting portion of GPRC5D includes at least one antigen-binding domain targeting GPRC5D. In some embodiments, the second targeting portion of GPRC5D includes at least one CDR in the variable region VH of the antibody heavy chain, said VH containing the amino acid sequence shown in SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87.

[0399] In some embodiments, the second targeting portion of GPRC5D includes HCDR3, and the HCDR3 comprises the amino acid sequences shown in SEQ ID NO:75, SEQ ID NO:78, and / or SEQ ID NO:81.

[0400] In some embodiments, the second targeting portion of GPRC5D includes HCDR2, and the HCDR2 comprises the amino acid sequences shown in SEQ ID NO:74, SEQ ID NO:77, and / or SEQ ID NO:80.

[0401] In some embodiments, the second targeting portion of GPRC5D includes HCDR1, and the HCDR1 contains the amino acid sequences shown in SEQ ID NO:73, SEQ ID NO:76, and / or SEQ ID NO:79.

[0402] In some embodiments, the second targeting portion of GPRC5D includes HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group shown below:

[0403] (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75;

[0404] (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and

[0405] (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:81.

[0406] In some embodiments, the second targeting portion of GPRC5D includes an antigen-binding domain that targets GPRC5D.

[0407] In some embodiments, the second targeting portion of GPRC5D includes two antigen-binding domains targeting GPRC5D: a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first and second antigen-binding domains target the same epitope of GPRC5D. In some embodiments, the first and second antigen-binding domains target different epitopes of GPRC5D. In some embodiments, the first and second antigen-binding domains targeting GPRC5D contain the same or different amino acid sequences.

[0408] In some embodiments, the first antigen-binding domain targeting GPRC5D and the second antigen-binding domain targeting GPRC5D are directly or indirectly linked. In some embodiments, the first antigen-binding domain targeting GPRC5D and the second antigen-binding domain targeting GPRC5D are linked by a linker. In one embodiment, the linker is a linker peptide. For example, the linker peptide contains the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linker peptide contains the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0409] In some embodiments, the first antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D comprise amino acid sequences selected from the group consisting of:

[0410] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0411] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0412] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81;

[0413] (4) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0414] (5) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0415] (6) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81;

[0416] (7) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0417] (8) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81, and the HCDR1 of the second antigen-binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; and

[0418] (9) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81.

[0419] In some embodiments, the second targeting portion of GPRC5D includes three antigen-binding domains targeting GPRC5D: a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain. In some embodiments, the first, second, and third antigen-binding domains target the same epitope of GPRC5D. In some embodiments, the first, second, and third antigen-binding domains target different epitopes of GPRC5D. In some embodiments, the first, second, and third antigen-binding domains targeting GPRC5D contain the same or different amino acid sequences.

[0420] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain targeting GPRC5D are directly or indirectly linked. In some embodiments, the first, second, and third antigen-binding domains targeting GPRC5D are linked by a linker. In one embodiment, the linker is a linker peptide. For example, the linker peptide contains the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linker peptide contains the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0421] For example, the first antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3; the second antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3; and the third antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D, the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D, and the HCDR1, HCDR2, and HCDR3 of the third antigen-binding domain targeting GPRC5D comprise amino acid sequences selected from the group consisting of:

[0422] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78; and the third antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81.

[0423] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; and the third antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0424] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78; the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; and the third antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81.

[0425] (4) The HCDR1 of the first antigen binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; the HCDR1 of the second antigen binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81; and the HCDR1 of the third antigen binding domain is SEQ ID NO:73, the HCDR2 is SEQ ID NO:74, and the HCDR3 is SEQ ID NO:75.

[0426] (5) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, HCDR2 is SEQ ID NO:80, and HCDR3 is SEQ ID NO:81; the HCDR1 of the second antigen-binding domain is SEQ ID NO:73, HCDR2 is SEQ ID NO:74, and HCDR3 is SEQ ID NO:75; and the HCDR1 of the third antigen-binding domain is SEQ ID NO:76, HCDR2 is SEQ ID NO:77, and HCDR3 is SEQ ID NO:78; and

[0427] (6) The first antigen-binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; the second antigen-binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78; and the third antigen-binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75.

[0428] In some embodiments, the first targeting portion targeting BCMA is directly or indirectly linked to the second targeting portion targeting GPRC5D. In some embodiments, the first targeting portion targeting BCMA and the second targeting portion targeting GPRC5D are linked by a linker. In some embodiments, the linker comprises a linker peptide. For example, the linker peptide comprises the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linker peptide comprises the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0429] In some embodiments, the first targeting portion of BCMA is connected to the second targeting portion of GPRC5D. For example, the first antigen-binding domain of the first targeting portion of BCMA is connected to the second targeting portion of GPRC5D, and the second targeting portion of GPRC5D is connected to the second antigen-binding domain of the first targeting portion of BCMA. Alternatively, the first antigen-binding domain of the first targeting portion of BCMA is connected to the second antigen-binding domain of the first targeting portion of BCMA, and the second antigen-binding domain of the first targeting portion of BCMA is connected to the second targeting portion of GPRC5D.

[0430] In this application, the chimeric antigen receptor further includes a third targeting portion that targets CD38. The third targeting portion includes at least one CDR in the variable region VH of the antibody heavy chain, the VH containing the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112, or SEQ ID NO:110.

[0431] In some embodiments, the third targeting portion of CD38 includes HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:102. In some embodiments, the third targeting portion of CD38 includes HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:90 or SEQ ID NO:93.

[0432] In some embodiments, the third targeting portion includes HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:96, or SEQ ID NO:100. In some embodiments, the third targeting portion includes HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99, or SEQ ID NO:97.

[0433] In some embodiments, the third targeting portion of CD38 includes HCDR1, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:101 or SEQ ID NO:88. In some embodiments, the third targeting portion of CD38 includes HCDR1, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:98, or SEQ ID NO:94.

[0434] In some embodiments, the third targeting portion of CD38 includes HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group shown below:

[0435] (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90;

[0436] (2) HCDR1: SEQ ID NO:101, HCDR2: SEQ ID NO:100, and HCDR3: SEQ ID NO:102; and

[0437] (3) HCDR1: SEQ ID NO: 101, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO: 102.

[0438] For example, the third targeting portion of CD38 includes HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group shown below:

[0439] (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90;

[0440] (2) HCDR1: SEQ ID NO:91, HCDR2: SEQ ID NO:92, and HCDR3: SEQ ID NO:93;

[0441] (3) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:95, and HCDR3: SEQ ID NO:93;

[0442] (4) HCDR1: SEQ ID NO:98, HCDR2: SEQ ID NO:99, and HCDR3: SEQ ID NO:93; and

[0443] (5) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:97, and HCDR3: SEQ ID NO:93.

[0444] In some embodiments, the first targeting portion targeting BCMA, the second targeting portion targeting GPRC5D, and the third targeting portion targeting CD38 are directly or indirectly linked. In some embodiments, the first targeting portion targeting BCMA, the second targeting portion targeting GPRC5D, and the third targeting portion targeting CD38 are linked by a linker. In some embodiments, the linker comprises a linker peptide. For example, the linker peptide comprises the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linker peptide comprises the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0445] In some embodiments, the first targeting portion of the BCMA target, the second targeting portion of the GPRC5D target, and the third targeting portion of the CD38 target are connected.

[0446] In some embodiments, the first targeting portion targeting BCMA, the second targeting portion targeting GPRC5D, and the third targeting portion targeting CD38 comprise an antibody or an antigen-binding fragment thereof.

[0447] In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0448] In some embodiments, the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0449] Fusion protein

[0450] On the other hand, this application provides a fusion protein comprising an extracellular region and a transmembrane region, wherein the extracellular region comprises at least one antigen-binding domain targeting BCMA, and the antigen-binding domain comprises the antigen-binding protein described in this application.

[0451] In some embodiments, the first targeting portion of BCMA includes an antigen-binding domain targeting BCMA. In some embodiments, the first targeting portion of BCMA includes HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0452] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0453] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0454] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0455] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0456] For example, the antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0457] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0458] (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2;

[0459] (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2;

[0460] (4) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 3;

[0461] (5) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 4;

[0462] (6) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 5;

[0463] (7) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 6

[0464] (8) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 7;

[0465] (9) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 8;

[0466] (10) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 9;

[0467] (11) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 10;

[0468] (12) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 11;

[0469] (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 12;

[0470] (14) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 13;

[0471] (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14;

[0472] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0473] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0474] For example, the first targeting portion targeting BCMA may be SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, or SEQ ID NO:72.

[0475] In some embodiments, the first targeting portion of BCMA includes a first antigen-binding domain targeting BCMA and a second antigen-binding domain targeting BCMA.

[0476] In some embodiments, the first antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and the first and second antigen-binding domains targeting BCMA are each independently selected from any one of the following amino acid sequences:

[0477] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0478] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0479] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0480] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0481] For example, the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA are each independently selected from any one of the following groups of amino acid sequences:

[0482] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0483] (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2;

[0484] (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2;

[0485] (4) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 3;

[0486] (5) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 4;

[0487] (6) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 5;

[0488] (7) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 6

[0489] (8) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 7;

[0490] (9) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 8;

[0491] (10) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 9;

[0492] (11) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 10;

[0493] (12) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 11;

[0494] (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 12;

[0495] (14) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 13;

[0496] (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14;

[0497] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0498] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0499] For example, the first antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain contain amino acid sequences selected from the group consisting of:

[0500] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0501] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0502] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0503] (4) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0504] (5) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0505] (6) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0506] (7) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0507] (8) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0508] (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0509] (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0510] (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0511] (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0512] (13) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0513] (14) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0514] (15) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0515] (16) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0516] (17) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0517] (18) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0518] (19) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0519] (20) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2.

[0520] (21) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2.

[0521] (22) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0522] (23) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0523] (24) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0524] (25) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0525] (26) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0526] (27) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0527] (28) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0528] (29) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13.

[0529] (30) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0530] (31) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0531] (32) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0532] (33) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2.

[0533] (34) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0534] (35) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; and

[0535] (36) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15.

[0536] For example, the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA can each be independently selected from the group consisting of: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0537] In this application, the transmembrane region of the fusion protein comprises a transmembrane domain derived from one or more proteins selected from the group consisting of: low-density lipoprotein receptor-associated protein or fragments thereof, CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0538] For example, the transmembrane region is a transmembrane region derived from CD8.

[0539] For example, the transmembrane region is a transmembrane region of a low-density lipoprotein receptor-associated protein or a fragment thereof. In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments. In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof. For example, the low-density lipoprotein receptor-associated protein or a fragment thereof is the Ori2 protein. For example, the low-density lipoprotein receptor-associated protein or a fragment thereof comprises the amino acid sequence shown in SEQ ID NO:124.

[0540] The fusion protein described in this application may further include a co-stimulatory domain, the co-stimulatory signaling region comprising an intracellular co-stimulatory signaling region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

[0541] For example, the co-stimulatory signal region is an intracellular co-stimulatory signal region derived from 4-1BB or CD28. For example, the co-stimulatory signal region may contain the amino acid sequence shown in SEQ ID NO:120 or SEQ ID NO:121.

[0542] In this application, the fusion protein can bind to a chimeric antigen receptor that targets GPRC5D.

[0543] In some embodiments, the chimeric antigen receptor targeting GPRC5D may include an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain includes at least one antigen-binding domain.

[0544] In some embodiments, the antigen-binding domain has an antigen-binding domain comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group shown below:

[0545] (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75;

[0546] (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and

[0547] (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:81.

[0548] In some embodiments, the extracellular domain of the chimeric antigen receptor targeting GPRC5D has two antigen-binding domains, a first antigen-binding domain and a second antigen-binding domain. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are each independently selected from the amino acid sequences shown in the following group:

[0549] (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75;

[0550] (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and

[0551] (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:81.

[0552] For example, the first antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D comprise amino acid sequences selected from the group consisting of:

[0553] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0554] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0555] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81;

[0556] (4) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0557] (5) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0558] (6) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81;

[0559] (7) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0560] (8) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81, and the HCDR1 of the second antigen-binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; and

[0561] (9) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81.

[0562] In some embodiments, the extracellular domain of the chimeric antigen receptor targeting GPRC5D has three antigen-binding domains: a first antigen-binding domain, a second antigen-binding domain, and a third antigen-binding domain. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and the third antigen-binding domain are each independently selected from the amino acid sequences shown in the following group:

[0563] (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75;

[0564] (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and

[0565] (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:81.

[0566] For example, the first antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3; the second antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3; and the third antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D contain amino acid sequences selected from the group consisting of:

[0567] (1) The HCDR1 of the first antigen binding domain is SEQ ID NO:73, the HCDR2 is SEQ ID NO:74, the HCDR3 is SEQ ID NO:75, the HCDR1 of the second antigen binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, the HCDR3 is SEQ ID NO:78, and the HCDR1 of the third antigen binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, the HCDR3 is SEQ ID NO:81;

[0568] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; and the third antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0569] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78; the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; and the third antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81.

[0570] (4) The HCDR1 of the first antigen binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; the HCDR1 of the second antigen binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81; and the HCDR1 of the third antigen binding domain is SEQ ID NO:73, the HCDR2 is SEQ ID NO:74, and the HCDR3 is SEQ ID NO:75.

[0571] (5) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, HCDR2 is SEQ ID NO:80, and HCDR3 is SEQ ID NO:81; the HCDR1 of the second antigen-binding domain is SEQ ID NO:73, HCDR2 is SEQ ID NO:74, and HCDR3 is SEQ ID NO:75; and the HCDR1 of the third antigen-binding domain is SEQ ID NO:76, HCDR2 is SEQ ID NO:77, and HCDR3 is SEQ ID NO:78; and

[0572] (6) The first antigen-binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; the second antigen-binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78; and the third antigen-binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75.

[0573] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain target the same epitope of GPRC5D. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain target different epitopes of GPRC5D.

[0574] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain contain the same or different amino acid sequences.

[0575] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain targeting GPRC5D are directly or indirectly linked. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain are linked by a linker. In one embodiment, the linker is a linking peptide. For example, the linking peptide comprises the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linking peptide comprises the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0576] In some embodiments, the transmembrane domain of the chimeric antigen receptor targeting GPRC5D may comprise a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, DAP10, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane domain may be the transmembrane domain of CD8.

[0577] In some embodiments, the intracellular domain of the chimeric antigen receptor targeting GPRC5D may include an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and other domains containing at least one ITAM. For example, the intracellular domain may be an intracellular domain derived from CD3ζ.

[0578] In this application, the fusion protein can bind to a chimeric antigen receptor targeting CD38.

[0579] In some embodiments, the chimeric antigen receptor targeting CD38 includes an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain including at least one antigen-binding domain. In some embodiments, the antigen-binding domain targeting CD38 includes at least one CDR in the variable region VH of the antibody heavy chain, the VH containing the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112, or SEQ ID NO:110.

[0580] In some embodiments, the antigen-binding domain targeting CD38 includes HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:102. In some embodiments, the antigen-binding domain targeting CD38 includes HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:90 or SEQ ID NO:93.

[0581] In some embodiments, the antigen-binding domain targeting CD38 includes HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:96, or SEQ ID NO:100. In some embodiments, the antigen-binding domain targeting CD38 includes HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99, or SEQ ID NO:97.

[0582] In some embodiments, the antigen-binding domain targeting CD38 includes HCDR1, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:101 or SEQ ID NO:88. In some embodiments, the antigen-binding domain targeting CD38 includes HCDR1, and HCDR1 comprises the amino acid sequence shown in SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:98, or SEQ ID NO:94.

[0583] In some embodiments, the antigen-binding domain targeting CD38 includes HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group shown below:

[0584] (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90;

[0585] (2) HCDR1: SEQ ID NO:101, HCDR2: SEQ ID NO:100, and HCDR3: SEQ ID NO:102; and

[0586] (3) HCDR1: SEQ ID NO: 101, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO: 102.

[0587] In some embodiments, the antigen-binding domain targeting CD38 includes HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group shown below:

[0588] (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90;

[0589] (2) HCDR1: SEQ ID NO:91, HCDR2: SEQ ID NO:92, and HCDR3: SEQ ID NO:93;

[0590] (3) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:95, and HCDR3: SEQ ID NO:93;

[0591] (4) HCDR1: SEQ ID NO:98, HCDR2: SEQ ID NO:99, and HCDR3: SEQ ID NO:93; and

[0592] (5) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:97, and HCDR3: SEQ ID NO:93.

[0593] In some embodiments, the transmembrane domain of the chimeric antigen receptor targeting CD38 includes a transmembrane domain comprising a protein derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. For example, the transmembrane region is a transmembrane region derived from CD8.

[0594] In some embodiments, the intracellular domain of the chimeric antigen receptor targeting CD38 comprises an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the intracellular signaling region is a signal transduction domain derived from CD3ζ.

[0595] In this application, the fusion protein covalently binds to a chimeric antigen receptor targeting CPRC5D and / or CD38. For example, the covalent binding is via a disulfide bond.

[0596] cell

[0597] On the other hand, this application provides a cell comprising and / or expressing a chimeric antigen receptor or fusion protein targeting B cell maturation antigen (BCMA), a chimeric antigen receptor targeting GPRC5D, and / or a fusion protein targeting CD38, wherein the chimeric antigen receptor or fusion protein targeting BCMA comprises the antigen-binding protein described in this application.

[0598] For example, the chimeric antigen receptor or fusion protein targeting BCMA contains at least one antigen-binding domain targeting BCMA.

[0599] For example, the chimeric antigen receptor or fusion protein targeting BCMA contains an antigen-binding domain that targets BCMA.

[0600] For example, the antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 contain any one of the following amino acid sequences:

[0601] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0602] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0603] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0604] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0605] For example, the antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 comprise any one of the following amino acid sequences:

[0606] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0607] (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2;

[0608] (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2;

[0609] (4) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 3;

[0610] (5) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 4;

[0611] (6) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 5;

[0612] (7) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 6

[0613] (8) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 7;

[0614] (9) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 8;

[0615] (10) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 9;

[0616] (11) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 10;

[0617] (12) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 11;

[0618] (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 12;

[0619] (14) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 13;

[0620] (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14;

[0621] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0622] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0623] For example, the antigen-binding domain targeting BCMA may be SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, or SEQ ID NO:72.

[0624] For example, the chimeric antigen receptor or fusion protein targeting BCMA includes two BCMA-targeting antigen-binding domains: a first antigen-binding domain targeting BCMA and a second antigen-binding domain targeting BCMA.

[0625] For example, the first antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and both the first and second antigen-binding domains targeting BCMA are independently selected from any one of the following groups of amino acid sequences:

[0626] (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16;

[0627] (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17;

[0628] (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and

[0629] (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0630] For example, the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA are each independently selected from any one of the following groups of amino acid sequences:

[0631] (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1;

[0632] (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2;

[0633] (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2;

[0634] (4) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 3;

[0635] (5) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:21, and HCDR3: SEQ ID NO:4;

[0636] (6) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:5;

[0637] (7) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:6

[0638] (8) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:7;

[0639] (9) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:8;

[0640] (10) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:9;

[0641] (11) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:22, and HCDR3: SEQ ID NO:10;

[0642] (12) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:11;

[0643] [[ID=二十五]](13) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:12;

[0644] (14) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:13;

[0645] (15) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:14;

[0646] (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and

[0647] (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15.

[0648] For example, the first antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting BCMA includes HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting BCMA and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting BCMA comprise amino acid sequences selected from the group consisting of:

[0649] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0650] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0651] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0652] (4) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0653] (5) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0654] (6) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0655] (7) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0656] (8) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0657] (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0658] (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0659] (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0660] (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0661] (13) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0662] (14) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0663] (15) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0664] (16) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0665] (17) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0666] (18) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0667] (19) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0668] (20) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2.

[0669] (21) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2.

[0670] (22) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0671] (23) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13;

[0672] (24) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0673] (25) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0674] (26) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0675] (27) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2;

[0676] (28) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0677] (29) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13.

[0678] (30) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15;

[0679] (31) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1;

[0680] (32) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2;

[0681] (33) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2.

[0682] (34) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11;

[0683] (35) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; and

[0684] (36) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15.

[0685] For example, the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA can each be independently selected from the group consisting of: SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:72.

[0686] In some embodiments, the chimeric antigen receptor targeting GPRC5D includes at least one antigen-binding domain targeting GPRC5D.

[0687] In some embodiments, the antigen-binding domain targeting GPRC5D includes an antigen-binding domain targeting GPRC5D.

[0688] In some embodiments, the antigen-binding domain targeting GPRC5D comprises two antigen-binding domains targeting GPRC5D: a first antigen-binding domain targeting GPRC5D and a second antigen-binding domain targeting GPRC5D.

[0689] In some embodiments, the antigen-binding domain targeting GPRC5D includes three antigen-binding domains targeting GPRC5D: a first antigen-binding domain targeting GPRC5D, a second antigen-binding domain targeting GPRC5D, and a third antigen-binding domain targeting GPRC5D.

[0690] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain targeting GPRC5D are each independently selected from the amino acid sequences shown in the group below:

[0691] (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75;

[0692] (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and

[0693] (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:81.

[0694] For example, the first antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D comprise amino acid sequences selected from the group consisting of:

[0695] (1) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0696] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0697] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81;

[0698] (4) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0699] (5) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0700] (6) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81;

[0701] (7) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75;

[0702] (8) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81, and the HCDR1 of the second antigen-binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; and

[0703] (9) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81.

[0704] For example, the first antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3; the second antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3; and the third antigen-binding domain targeting GPRC5D includes HCDR1, HCDR2, and HCDR3. The HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D, the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D, and the HCDR1, HCDR2, and HCDR3 of the third antigen-binding domain targeting GPRC5D comprise amino acid sequences selected from the group consisting of:

[0705] (1) The HCDR1 of the first antigen binding domain is SEQ ID NO:73, the HCDR2 is SEQ ID NO:74, the HCDR3 is SEQ ID NO:75, the HCDR1 of the second antigen binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, the HCDR3 is SEQ ID NO:78, and the HCDR1 of the third antigen binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, the HCDR3 is SEQ ID NO:81;

[0706] (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; and the third antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78.

[0707] (3) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78; the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; and the third antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81.

[0708] (4) The HCDR1 of the first antigen binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; the HCDR1 of the second antigen binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81; and the HCDR1 of the third antigen binding domain is SEQ ID NO:73, the HCDR2 is SEQ ID NO:74, and the HCDR3 is SEQ ID NO:75.

[0709] (5) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, HCDR2 is SEQ ID NO:80, and HCDR3 is SEQ ID NO:81; the HCDR1 of the second antigen-binding domain is SEQ ID NO:73, HCDR2 is SEQ ID NO:74, and HCDR3 is SEQ ID NO:75; and the HCDR1 of the third antigen-binding domain is SEQ ID NO:76, HCDR2 is SEQ ID NO:77, and HCDR3 is SEQ ID NO:78; and

[0710] (6) The first antigen-binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; the second antigen-binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78; and the third antigen-binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75.

[0711] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain targeting GPRC5D target the same or different epitopes of GPRC5D.

[0712] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain targeting GPRC5D contain the same or different amino acid sequences.

[0713] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain targeting GPRC5D are directly or indirectly linked. In some embodiments, the first antigen-binding domain, the second antigen-binding domain, and / or the third antigen-binding domain targeting GPRC5D are linked by a linker. For example, the linker is a linker peptide. For example, the linker peptide contains the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linker peptide contains the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0714] In some embodiments, the CD38-targeting fusion protein includes an antigen-binding domain that targets CD38.

[0715] In some embodiments, the antigen-binding domain targeting CD38 includes HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group shown below:

[0716] (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90;

[0717] (2) HCDR1: SEQ ID NO:101, HCDR2: SEQ ID NO:100, and HCDR3: SEQ ID NO:102; and

[0718] (3) HCDR1: SEQ ID NO: 101, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO: 102.

[0719] In this application, the cell may further include a hinge region. In some embodiments, the hinge region may comprise a hinge region of one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0720] For example, the hinge area is a hinge area derived from CD8 or CD28.

[0721] In this application, the cell may further include a transmembrane region. In some embodiments, the transmembrane region comprises a transmembrane domain derived from one or more proteins selected from the group consisting of: low-density lipoprotein receptor-associated protein or fragments thereof, CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0722] For example, the transmembrane region is the transmembrane region of CD8, CD28, and / or low-density lipoprotein receptor-associated protein or fragments thereof.

[0723] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments. In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

[0724] For example, the low-density lipoprotein receptor-associated protein or a fragment thereof is the Ori2 protein.

[0725] For example, the transmembrane region contains the amino acid sequences shown in SEQ ID NO:118, SEQ ID NO:119 and / or SEQ ID NO:124.

[0726] In this application, the cell may further include a co-stimulatory signaling region. In some embodiments, the co-stimulatory signaling region comprises an intracellular co-stimulatory signaling region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. For example, the co-stimulatory signaling region is an intracellular co-stimulatory signaling region derived from 4-1BB and / or CD28. For example, the co-stimulatory signal region contains the amino acid sequences shown in SEQ ID NO:120 and / or SEQ ID NO:121.

[0727] In this application, the cell may further include an intracellular domain. In some embodiments, the intracellular domain comprises an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. For example, the intracellular domain is an intracellular signaling region derived from CD3ζ. For example, the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:122.

[0728] In this application, the cell may include BCMACAR.

[0729] In some embodiments, the BCMACAR includes an extracellular domain, the aforementioned hinge region, the aforementioned transmembrane region, the aforementioned intracellular co-stimulatory signaling region, and the aforementioned intracellular signaling region. In some embodiments, the extracellular domain may contain any one, two, or three of the aforementioned BCMA antigen-binding domains.

[0730] For example, in the cells provided in this application, BCMACAR from the N-terminus to the C-terminus can be: an antigen-binding domain targeting BCMA, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ.

[0731] For example, in the cells provided in this application, BCMACAR from the N-terminus to the C-terminus can be: a first antigen-binding domain targeting BCMA, a second antigen-binding domain targeting BCMA, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ.

[0732] In this application, the cells may include BCMA×GPRC5D bispecific CAR.

[0733] In some embodiments, the BCMA×GPRC5D bispecific CAR includes an extracellular domain, the aforementioned hinge region, the aforementioned transmembrane region, the aforementioned intracellular co-stimulatory signaling region, and the aforementioned intracellular signaling region. In some embodiments, the extracellular domain may contain any one, two, or three of the aforementioned BCMA antigen-binding domains and any one, two, or three of the aforementioned GPRC5D antigen-binding domains.

[0734] In some embodiments, the BCMA×GPRC5D bispecific CAR includes an extracellular domain, the aforementioned transmembrane region, the aforementioned intracellular co-stimulatory signaling region, the aforementioned intracellular signaling region, and a low-density lipoprotein receptor-associated protein or a fragment thereof. In some embodiments, the extracellular domain may contain any one, two, or three of the aforementioned BCMA antigen-binding domains and any one, two, or three of the aforementioned GPRC5D antigen-binding domains.

[0735] For example, in the cells provided in this application, the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus can be: an antigen-binding domain targeting GPRC5D, a first antigen-binding domain targeting BCMA, a second antigen-binding domain targeting BCMA, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ, and the CD38 fusion protein from the N-terminus to the C-terminus can be: an antigen-binding domain targeting CD38 and an Ori2 transmembrane domain.

[0736] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:83, SEQ ID NO:42, and SEQ ID NO:70.

[0737] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:83, SEQ ID NO:70, and SEQ ID NO:42.

[0738] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:42, SEQ ID NO:70, and SEQ ID NO:83.

[0739] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70, SEQ ID NO:42, and SEQ ID NO:83.

[0740] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70, SEQ ID NO:83, and SEQ ID NO:42.

[0741] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:42, SEQ ID NO:83, and SEQ ID NO:70.

[0742] In this application, the cells may include BCMA×GPRC5D bispecific CAR and CD38 fusion protein.

[0743] In some embodiments, the BCMA×GPRC5D bispecific CAR includes an extracellular domain, the aforementioned hinge region, the aforementioned transmembrane region, the aforementioned intracellular co-stimulatory signaling region, and the aforementioned intracellular signaling region. In some embodiments, the extracellular domain may contain any one, two, or three of the aforementioned BCMA antigen-binding domains and any one, two, or three of the aforementioned GPRC5D antigen-binding domains.

[0744] In some embodiments, the CD38 fusion protein includes an antigen-binding domain targeting CD38 and a transmembrane domain. In some embodiments, the CD38 fusion protein includes an antigen-binding domain targeting CD38, the aforementioned transmembrane domain, and the aforementioned co-stimulatory signaling region. In some embodiments, the CD38 fusion protein includes an antigen-binding domain targeting CD38, the aforementioned hinge region, the aforementioned transmembrane domain, and the aforementioned co-stimulatory signaling region.

[0745] For example, the antigen-binding domain targeting CD38 may comprise the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112 or SEQ ID NO:110.

[0746] In some embodiments, the BCMA×GPRC5D bispecific CAR can be, from the N-terminus to the C-terminus, an extracellular domain, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ. The CD38 fusion protein can be, from the N-terminus to the C-terminus, an antigen-binding domain targeting CD38 and an Ori2 transmembrane domain.

[0747] In some embodiments, the BCMA×GPRC5D bispecific CAR can be, from the N-terminus to the C-terminus, an extracellular domain, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ. The CD38 fusion protein can be, from the N-terminus to the C-terminus, an antigen-binding domain targeting CD38, a transmembrane domain of CD8, and a co-stimulatory signaling region of CD28.

[0748] In some embodiments, the BCMA×GPRC5D bispecific CAR can be, from the N-terminus to the C-terminus, an extracellular domain, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ. The CD38 fusion protein can be, from the N-terminus to the C-terminus, an antigen-binding domain targeting CD38, a hinge region of CD28, a transmembrane domain of CD8, and a co-stimulatory signaling region of CD28.

[0749] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:83, SEQ ID NO:42, and SEQ ID NO:70.

[0750] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:83, SEQ ID NO:70, and SEQ ID NO:42.

[0751] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:42, SEQ ID NO:70, and SEQ ID NO:83.

[0752] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70, SEQ ID NO:42, and SEQ ID NO:83.

[0753] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70, SEQ ID NO:83, and SEQ ID NO:42.

[0754] For example, in the cells of this application, the extracellular domain of the BCMA×GPRC5D bispecific CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:42, SEQ ID NO:83, and SEQ ID NO:70.

[0755] In this application, the cells may include BCMA fusion protein and GPRC5D CAR.

[0756] In some embodiments, the BCMA fusion protein may be, from the N-terminus to the C-terminus, an extracellular domain targeting BCMA, the aforementioned transmembrane domain, and the aforementioned co-stimulatory signaling region.

[0757] In some embodiments, the extracellular domain targeting BCMA may include any one, two, or three of the aforementioned BCMA antigen-binding domains.

[0758] In some embodiments, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, the aforementioned transmembrane domain, and the aforementioned intracellular signaling region. In some embodiments, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, the aforementioned hinge region, the aforementioned transmembrane domain, and the aforementioned intracellular signaling region. In some embodiments, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, the aforementioned transmembrane domain, the aforementioned co-stimulatory signaling region, and the aforementioned intracellular signaling region. For example, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, a hinge region of CD8, a transmembrane region of CD8, and an intracellular signaling region of CD3ζ. For example, the GPRC5D CAR, from its N-terminus to its C-terminus, can be: an extracellular domain targeting GPRC5D, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of CD28, and an intracellular signaling region of CD3ζ. Alternatively, the GPRC5D CAR, from its N-terminus to its C-terminus, can be: an extracellular domain targeting GPRC5D, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ.

[0759] In some embodiments, the extracellular domain targeting GPRC5D may include any one, two, or three of the aforementioned GPRC5D antigen-binding domains.

[0760] In some embodiments, the BCMA fusion protein and the GPRC5D CAR are covalently linked. For example, the BCMA fusion protein and the GPRC5D CAR are linked by disulfide bonds.

[0761] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein may contain the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, or SEQ ID NO:72, and the extracellular domain of the GPRC5D CAR may contain the amino acid sequences shown in SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87.

[0762] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein from the N-terminus to the C-terminus may contain the amino acid sequences shown below: SEQ ID NO:42 and SEQ ID NO:70, and the extracellular domain of the GPRC5D CAR may contain the amino acid sequences shown in SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87.

[0763] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70 and SEQ ID NO:42, and the extracellular domain of the GPRC5D CAR may contain the amino acid sequences shown in SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87.

[0764] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70 and SEQ ID NO:42, and the extracellular domain of the GPRC5D CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:85 and SEQ ID NO:87.

[0765] In some embodiments, the cell may also include low-density lipoprotein receptor-associated protein or fragments thereof.

[0766] In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments. In some embodiments, the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

[0767] For example, the low-density lipoprotein receptor-associated protein or a fragment thereof is the Ori2 protein.

[0768] In this application, the cells may include BCMA fusion protein, GPRC5D CAR and CD38 fusion protein.

[0769] In some embodiments, the BCMA fusion protein may be, from the N-terminus to the C-terminus, an extracellular domain targeting BCMA, the aforementioned transmembrane domain, and the aforementioned co-stimulatory signaling region.

[0770] In some embodiments, the extracellular domain targeting BCMA may include any one, two, or three of the aforementioned BCMA antigen-binding domains.

[0771] In some embodiments, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, the aforementioned transmembrane domain, and the aforementioned intracellular signaling region. In some embodiments, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, the aforementioned hinge region, the aforementioned transmembrane domain, and the aforementioned intracellular signaling region. In some embodiments, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, the aforementioned transmembrane domain, the aforementioned co-stimulatory signaling region, and the aforementioned intracellular signaling region. For example, the GPRC5D CAR from the N-terminus to the C-terminus may comprise: an extracellular domain targeting GPRC5D, a hinge region of CD8, a transmembrane region of CD8, and an intracellular signaling region of CD3ζ. For example, the GPRC5D CAR, from its N-terminus to its C-terminus, can be: an extracellular domain targeting GPRC5D, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of CD28, and an intracellular signaling region of CD3ζ. Alternatively, the GPRC5D CAR, from its N-terminus to its C-terminus, can be: an extracellular domain targeting GPRC5D, a hinge region of CD8, a transmembrane region of CD8, an intracellular co-stimulatory signaling region of 4-1BB, and an intracellular signaling region of CD3ζ.

[0772] In some embodiments, the extracellular domain targeting GPRC5D may include any one, two, or three of the aforementioned GPRC5D antigen-binding domains.

[0773] In some embodiments, the CD38 fusion protein, from its N-terminus to its C-terminus, may comprise: an antigen-binding domain targeting CD38, the aforementioned hinge region, and the aforementioned transmembrane domain. In some embodiments, the CD38 fusion protein, from its N-terminus to its C-terminus, may comprise: an antigen-binding domain targeting CD38, the aforementioned hinge region, the aforementioned transmembrane domain, and the aforementioned co-stimulatory signaling region. For example, the CD38 fusion protein, from its N-terminus to its C-terminus, may comprise: an antigen-binding domain targeting CD38, a hinge region of CD28, and a transmembrane domain of CD28. For example, the CD38 fusion protein, from its N-terminus to its C-terminus, may comprise: an antigen-binding domain targeting CD38, a hinge region of CD28, a transmembrane domain of CD28, and a co-stimulatory signaling region of 4-1BB. For example, the CD38 fusion protein, from its N-terminus to its C-terminus, may comprise: an antigen-binding domain targeting CD38, a hinge region of CD28, a transmembrane domain of CD28, and a co-stimulatory signaling region of 4-1BB. For example, the CD38 fusion protein can be, from the N-terminus to the C-terminus, an antigen-binding domain targeting CD38, a hinge region of CD28, a transmembrane domain of CD28, and a co-stimulatory signaling region of CD28.

[0774] For example, the antigen-binding domain targeting CD38 may comprise the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112 or SEQ ID NO:110.

[0775] In some embodiments, the BCMA fusion protein and the GPRC5D CAR are covalently linked. For example, the BCMA fusion protein and the GPRC5D CAR are linked by disulfide bonds.

[0776] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein may contain the amino acid sequences shown in SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, or SEQ ID NO:72, and the extracellular domain of the GPRC5D CAR may contain the amino acid sequences shown in SEQ ID NO:83, SEQ ID NO:85, or SEQ ID NO:87.

[0777] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein from the N-terminus to the C-terminus may contain the amino acid sequences shown below: SEQ ID NO:42 and SEQ ID NO:70, and the extracellular domain of the GPRC5D CAR may contain the amino acid sequences shown in SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87.

[0778] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70 and SEQ ID NO:42, and the extracellular domain of the GPRC5D CAR may contain the amino acid sequences shown in SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:87.

[0779] For example, in the cells of this application, the extracellular domain of the BCMA fusion protein from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:70 and SEQ ID NO:42, and the extracellular domain of the GPRC5D CAR from the N-terminus to the C-terminus may contain the following amino acid sequences: SEQ ID NO:85 and SEQ ID NO:87.

[0780] In this application, the cells comprise immune effector cells. In some embodiments, the cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells, and / or kinetic stem cells, or variants thereof. For example, the cells are T cells.

[0781] Nucleic acid molecules

[0782] On the other hand, this application provides an isolated nucleic acid molecule encoding the antigen-binding protein, the chimeric antigen receptor or fusion protein targeting BCMA, the chimeric antigen receptor targeting GPRC5D, and / or the fusion protein targeting CD38.

[0783] In this application, the nucleic acid sequence encoding the chimeric antigen receptor targeting BCMA includes the nucleic acid sequence encoding the aforementioned antigen-binding domain targeting BCMA. In some embodiments, the antigen-binding domain targeting BCMA comprises at least one CDR in VH, and the nucleic acid sequence encoding VH comprises one or more nucleotide sequences selected from the group consisting of: SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71.

[0784] In some embodiments, the antigen-binding domain targeting BCMA includes at least one of the aforementioned antigen-binding domains targeting BCMA.

[0785] In some embodiments, the antigen-binding domain targeting BCMA includes one of the aforementioned antigen-binding domains targeting BCMA.

[0786] In some embodiments, the antigen-binding domain targeting BCMA comprises two of the aforementioned antigen-binding domains targeting BCMA.

[0787] In some embodiments, the nucleic acid sequence encoding the aforementioned antigen-binding domain targeting BCMA comprises one or more nucleotide sequences selected from the group consisting of: SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, and SEQ ID NO:71.

[0788] In this application, the nucleic acid sequence encoding the chimeric antigen receptor targeting GPRC5D includes a nucleic acid sequence encoding the aforementioned antigen-binding domain targeting GPRC5D. In some embodiments, the antigen-binding domain targeting GPRC5D comprises at least one CDR in VH, and the nucleic acid sequence encoding the VH comprises the nucleotide sequence shown in SEQ ID NO:82, SEQ ID NO:84, or SEQ ID NO:86.

[0789] In some embodiments, the antigen-binding domain targeting GPRC5D includes at least one of the aforementioned antigen-binding domains targeting GPRC5D.

[0790] In some embodiments, the antigen-binding domain targeting GPRC5D includes one of the aforementioned antigen-binding domains targeting GPRC5D.

[0791] In some embodiments, the antigen-binding domain targeting GPRC5D comprises two of the aforementioned antigen-binding domains targeting GPRC5D.

[0792] In some embodiments, the antigen-binding domain targeting GPRC5D comprises three of the aforementioned antigen-binding domains targeting GPRC5D.

[0793] In some embodiments, the nucleic acid sequence encoding the aforementioned antigen-binding domain targeting GPRC5D comprises one or more nucleotide sequences selected from the group consisting of the nucleotide sequences shown in SEQ ID NO:82, SEQ ID NO:84, or SEQ ID NO:86.

[0794] In this application, the nucleic acid sequence encoding the fusion protein targeting CD38 includes a nucleic acid sequence encoding an antigen-binding domain targeting CD38. In some embodiments, the antigen-binding domain targeting CD38 comprises at least one CDR in VH, and the nucleic acid sequence encoding the VH comprises the nucleotide sequence shown in SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:111, or SEQ ID NO:109.

[0795] In some embodiments, the antigen-binding domain encoding CD38 comprises the nucleotide sequence shown in SEQ ID NO:103, SEQ ID NO:105 or SEQ ID NO:109.

[0796] In some embodiments, the nucleic acid molecule includes a nucleic acid sequence encoding the antigen-binding domain targeting BCMA, a nucleic acid sequence encoding the antigen-binding domain targeting GPRC5D, and / or a nucleic acid sequence encoding the antigen-binding domain targeting CD38.

[0797] In this application, the nucleic acid molecule further includes a nucleic acid sequence encoding a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88. In some embodiments, the co-stimulatory signaling domain is an intracellular co-stimulatory signaling domain derived from 4-1BB or CD28.

[0798] In this application, the nucleic acid molecule further includes a nucleic acid sequence encoding an intracellular domain, said intracellular domain comprising an intracellular domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM. In some embodiments, the intracellular domain is an intracellular domain derived from CD3ζ.

[0799] In this application, the nucleic acid molecule further includes a nucleic acid sequence encoding a transmembrane domain, the transmembrane domain comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD8 or CD28.

[0800] In this application, the nucleic acid molecule further includes a nucleic acid sequence encoding a hinge region, the hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT. In some embodiments, the hinge region is a hinge region derived from CD8 or CD28.

[0801] In this application, the nucleic acid molecule further includes a nucleic acid sequence encoding a cleavage peptide, wherein the cleavage peptide is selected from 2A peptides.

[0802] In some embodiments, the 2A peptide is selected from the group consisting of P2A, T2A, F2A, and E2A. For example, the 2A peptide comprises the amino acid sequence shown in SEQ ID NO:123.

[0803] In some embodiments, the nucleic acid molecule further includes a nucleic acid sequence encoding a linker. In some embodiments, the linker is a linker peptide. For example, the linker peptide comprises the amino acid sequence (GGGGS)n, where n is any positive integer from 1 to 10. For example, the linker peptide comprises the amino acid sequence (EAAAK)n, where n is any positive integer from 1 to 10.

[0804] In this application, the nucleic acid molecule further includes a nucleic acid sequence encoding a low-density lipoprotein receptor-associated protein (LDL-AAP) or a fragment thereof. In some embodiments, the LDL-AAP or a fragment thereof comprises one or more of the following: LDL-AAP 1-12 and a functional fragment thereof. In some embodiments, the LDL-AAP or a fragment thereof is LDL-AAP 5 and / or 6 or a fragment thereof. For example, the LDL-AAP or a fragment thereof is the Ori2 protein.

[0805] In this application, the nucleic acid molecule further includes a nucleic acid sequence encoding a signal peptide, wherein the signal peptide is derived from a CD8 signal peptide.

[0806] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target BCMA antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0807] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0808] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding the CD8 signal peptide; the nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; and a nucleic acid sequence encoding... The nucleic acid sequence encoding the CD8 or CD28 hinge region, the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, the nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain, the nucleic acid sequence encoding the CD3ζ intracellular domain, the nucleic acid sequence encoding the 2A peptide, the nucleic acid sequence encoding the CD8 signal peptide, the nucleic acid sequence encoding the target CD38 antigen-binding domain, the nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10), and the nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0809] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding the CD8 signal peptide; the nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; and a sequence encoding C... The nucleic acid sequence encoding the D8 or CD28 hinge region, the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, the nucleic acid sequence encoding the co-stimulatory signal domain of 4-1BB or CD28, the nucleic acid sequence encoding the CD3ζ intracellular domain, the nucleic acid sequence encoding the 2A peptide, the nucleic acid sequence encoding the CD8 signal peptide, the nucleic acid sequence encoding the target CD38 antigen-binding domain, the nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10), and the nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0810] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); the nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; and a nucleic acid sequence encoding... The nucleic acid sequence encoding the CD8 or CD28 hinge region, the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, the nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain, the nucleic acid sequence encoding the CD3ζ intracellular domain, the nucleic acid sequence encoding the 2A peptide, the nucleic acid sequence encoding the CD8 signal peptide, the nucleic acid sequence encoding the target CD38 antigen-binding domain, the nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10), and the nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0811] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; a nucleic acid sequence encoding a 2A peptide; a nucleic acid sequence encoding the CD8 signal peptide; the nucleic acid sequence encoding the target BCMA first antigen-binding domain; and (GGGGS)n (where n is 1). Nucleic acid sequences (any positive integer in -10), encoding the nucleic acid sequence targeting the second antigen-binding domain of BCMA, encoding the CD8 or CD28 hinge region, encoding the CD8 or CD28 transmembrane domain, encoding the 4-1BB or CD28 co-stimulatory signal domain, encoding the 2A peptide, encoding the CD8 signal peptide, encoding the nucleic acid sequence targeting the CD38 antigen-binding domain, encoding the CD8 or CD28 hinge region, and encoding the CD8 or CD28 transmembrane domain.

[0812] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; a nucleic acid sequence encoding a 2A peptide; a nucleic acid sequence encoding the CD8 signal peptide; the nucleic acid sequence encoding the target BCMA first antigen-binding domain; and a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10). The sequences include: a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain; a nucleic acid sequence encoding the 2A peptide; a nucleic acid sequence encoding the CD8 signal peptide; a nucleic acid sequence encoding the target CD38 antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; and a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain.

[0813] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; and a CD8 or CD28 hinge. The nucleic acid sequence of the region, the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, the nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain, the nucleic acid sequence encoding the CD3ζ intracellular domain, the nucleic acid sequence encoding the 2A peptide, the nucleic acid sequence encoding the CD8 signal peptide, the nucleic acid sequence encoding the target CD38 antigen-binding domain, the nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10), the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, and the nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain.

[0814] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding the CD8 signal peptide; the nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; and a sequence encoding C. Nucleic acid sequences encoding the D8 or CD28 hinge region, nucleic acid sequences encoding the CD8 or CD28 transmembrane domain, nucleic acid sequences encoding the 4-1BB or CD28 co-stimulatory signaling domain, nucleic acid sequences encoding the CD3ζ intracellular domain, nucleic acid sequences encoding the 2A peptide, nucleic acid sequences encoding the CD8 signal peptide, nucleic acid sequences encoding the target CD38 antigen-binding domain, nucleic acid sequences encoding the CD8 or CD28 hinge region, nucleic acid sequences encoding the CD8 or CD28 transmembrane domain, and nucleic acid sequences encoding the 4-1BB or CD28 co-stimulatory signaling domain.

[0815] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the first antigen-binding domain of GPRC5D; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the second antigen-binding domain of GPRC5D; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; and a nucleic acid sequence encoding the 2A peptide. The nucleic acid sequence encoding the CD8 signal peptide, the nucleic acid sequence encoding the target BCMA first antigen-binding domain, the nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10), the nucleic acid sequence encoding the target BCMA second antigen-binding domain, the nucleic acid sequence encoding the CD8 or CD28 hinge region, the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, the nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain, the nucleic acid sequence encoding the 2A peptide, and the nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0816] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target GPRC5D first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D second antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; a nucleic acid sequence encoding the 2A peptide; a nucleic acid sequence encoding the CD8 signal peptide; and a nucleic acid sequence encoding the target GPRC5D second antigen-binding domain. The nucleic acid sequence of the antigen-binding domain, the nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10), the nucleic acid sequence encoding the target BCMA second antigen-binding domain, the nucleic acid sequence encoding the CD8 or CD28 hinge region, the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, the nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain, the nucleic acid sequence encoding the 2A peptide, the nucleic acid sequence encoding the CD8 signal peptide, the nucleic acid sequence encoding the target CD38 antigen-binding domain, the nucleic acid sequence encoding the CD8 or CD28 hinge region, and the nucleic acid sequence encoding the CD8 or CD28 transmembrane domain.

[0817] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target GPRC5D first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D second antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; a nucleic acid sequence encoding the 2A peptide; a nucleic acid sequence encoding the CD8 signal peptide; and a nucleic acid sequence encoding the target BCMA first antigen-binding domain. The sequence includes a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10), a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the CD8 or CD28 hinge region, a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signaling domain, a nucleic acid sequence encoding the 2A peptide, a nucleic acid sequence encoding the CD8 signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the CD8 or CD28 hinge region, a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain, and a co-stimulatory signaling domain encoding the 4-1BB or CD28.

[0818] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); the nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; a nucleic acid sequence encoding the 2A peptide; and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0819] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; a nucleic acid sequence encoding the 2A peptide; and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0820] In some embodiments, the nucleic acid molecule comprises, from its 5' end to its 3' end, the following sequences: a nucleic acid sequence encoding a CD8 signal peptide; a nucleic acid sequence encoding the target BCMA first antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target GPRC5D antigen-binding domain; a nucleic acid sequence encoding (GGGGS)n (where n is any positive integer from 1 to 10); a nucleic acid sequence encoding the target BCMA second antigen-binding domain; a nucleic acid sequence encoding the CD8 or CD28 hinge region; a nucleic acid sequence encoding the CD8 or CD28 transmembrane domain; a nucleic acid sequence encoding the 4-1BB or CD28 co-stimulatory signal domain; a nucleic acid sequence encoding the CD3ζ intracellular domain; a nucleic acid sequence encoding the 2A peptide; and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

[0821] In this application, the nucleic acid molecule may be a nucleotide, deoxynucleotide, and / or ribonucleotide of any length in a separable form, which may encode the separable antigen-binding protein, the chimeric antigen receptor, and / or the fusion protein.

[0822] In this application, the nucleic acid molecule may further include a promoter. For example, the promoter may be a constitutive promoter. For example, the promoter may be the EF1α promoter.

[0823] carrier

[0824] On the other hand, this application also provides a vector that may include the aforementioned nucleic acid molecule. The vector can transform, transduce, or transfect host cells, causing its carried genetic material elements to be expressed within the host cells. For example, the vector may include a promoter, transcript, enhancer, replicon, selection element, and reporter gene. For example, the vector may include components that facilitate entry into the cell. To enable the nucleic acid molecule to replicate in the vector, the 5' and 3' ends of the nucleic acid molecule may also include long terminal repeat sequences.

[0825] For example, the vector can be a viral vector. For example, the vector can be a lentiviral vector.

[0826] Pharmaceutical Composition

[0827] On the other hand, this application also provides a pharmaceutical composition comprising the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the nucleic acid molecule, the carrier, and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0828] In some embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention may include liquid, freeze-dried, and lyophilized compositions.

[0829] In some embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delay agents that are compatible with drug administration, are generally safe and non-toxic, and are neither biologically nor otherwise undesirable.

[0830] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally.

[0831] Methods and uses

[0832] On the other hand, this application provides a method for preparing modified immune cells, which includes introducing the nucleic acid molecule and / or the carrier into immune effector cells.

[0833] On the other hand, this application provides a method for preventing, treating and / or alleviating diseases and / or symptoms associated with BCMA, GPRC5D and / or CD38 expression, the method comprising administering to a subject in need the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition.

[0834] On the other hand, this application also provides the use of the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition in the preparation of a medicament that can be used to prevent, alleviate and / or treat diseases and / or conditions associated with BCMA, GPRC5D and / or CD38 expression.

[0835] On the other hand, this application also provides the antigen-binding protein, the chimeric antigen receptor, the fusion protein, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition thereof for the prevention, relief and / or treatment of diseases and / or conditions associated with BCMA, GPRC5D and / or CD38 expression.

[0836] In some implementations, the diseases and / or conditions associated with BCMA, GPRC5D, and / or CD38 expression include tumors and / or autoimmune diseases.

[0837] In some implementations, the disease and / or condition includes solid tumors.

[0838] In some implementations, the disease and / or condition includes non-solid tumors.

[0839] In some implementations, the disease and / or condition includes hematologic malignancies and / or lymphomas.

[0840] In some implementations, the disease and / or condition includes myeloma.

[0841] Without being limited by any theory, the embodiments described below are merely for illustrating the various technical solutions of the present invention and are not intended to limit the scope of the present invention.

[0842] Example

[0843] Example 1. Screening of anti-BCMA antibodies using the artificially synthesized nanobody library NanoOri_1.0

[0844] By comparing 296 nanobodies and their structures without repeating sequences in the Protein Data Bank (PDB) database, the CDR mutation strategy was determined, with three possible CDR 3 amino acid lengths: 14, 17, and 21. Using the commercially available nanobody Caplacizumab as the backbone, the Caplacizumab nucleotide sequence was synthesized from its entire genome and cloned into the HP153 phage vector. Single-stranded DNA was then extracted from HP153, and the CDRs of the nanobodies were mutated using the Kunkel Mutagenesis method to obtain double-stranded DNA. The double-stranded DNA was electroporated into competent *E. coli* SS320 cells pre-infected with M13KO7 helper phage. After overnight culture, the phage supernatant was collected, ultimately constructing a strain with a diversity of 1.44 x 10⁻⁶. 10 The artificially synthesized nanobody library NanoOri_1.0 was used for subsequent antibody sequence screening.

[0845] As shown in Figure 1, select BCMA. ECD - His recombinant protein (constructed using the Yuanqi Biomolecular Cloning Platform; the amino acid sequence of human BCMA protein is shown in the Uniprot identifier: Q02223-1; the amino acid sequence of His is HHHHHH) was used in a series of four rounds of panning to synthesize a nanobody library. In short, the BCMA antigen was coated in PBS buffer using an ELISA tube. ECD- Incubate 1 mL of His protein overnight at 4°C; the next day, block the immunoassay tubes with PBS buffer containing 5% skim milk powder; add the room-temperature blocked phage to the immunoassay tubes and incubate at room temperature; wash with PBST (0.05% TWEEN-20); elute with Gly-HCl buffer, neutralize with Tris-HCl buffer; add to 20 mL of TG1 with an OD of approximately 0.8 in the logarithmic growth phase, mix well, and incubate at 37°C for 1 h; take 1 mL of bacterial culture to determine the phage titer and glycerol preservation; add helper phage, mix well, and incubate at 37°C for 1 h; add IPTG and Kans antibiotic, and incubate overnight at 30°C with shaking at 250 rpm; collect the supernatant, precipitate the phage with PEG / NaCl solution, and resuspend in PBS buffer; the resuspended phage is used for the next round of panning. As shown in Figure 2, after 4 rounds of panning, polyclonal ELISA showed significant enrichment of specific sequences. Identification of panned phage antibody clones using monoclonal ELISA: Human BCMA was coated onto 96-well ELISA plates. ECD -His protein, concentration 1 μg / mL, incubated overnight at 4℃. Non-specific binding sites were blocked with 5% skim milk powder. After thorough washing, the supernatant of a single phage clone was added to a 96-well plate and incubated at 37℃ for 2 h. After thorough washing, Anti-M13-HRP (GE healtcare, 27-9421-01) was added, and the plate was incubated at 37℃ for 45 min. After thorough washing, TMB was added for color development, and the reaction was incubated at room temperature for 5-10 min. Finally, the reaction was terminated with sulfuric acid, and the OD value of each well was measured at 450 nm.

[0846] As shown in Figure 3, Tables 1 and 2, 108 phage antibody clones specifically binding to human BCMA were obtained by single-clone ELISA. After sequencing, the VHH gene sequence was obtained. Based on sequence repeat frequency and CDR3 classification, 17 phage antibody clones were selected for further verification (the BCMA nanobodies isolated in this application), named NanoBCMA1.0, NanoBCMA3.2, NanoBCMA9.2, NanoBCMA11, NanoBCMA29, NanoBCMA32, NanoBCMA37, NanoBCMA40, NanoBCMA41, NanoBCMA42, NanoBCMA44, NanoBCMA45, NanoBCMA46, NanoBCMA47, NanoBCMA48, NanoBCMA49, and NanoBCMA54.

[0847] Table 1. Specific Monoclonal Positive Rate of Monoclonal ELISA

[0848] Table 2. CDR sequences of 17 specific clones (based on IMGT classification)

[0849] Example 2. Targeting BCMA V H Construction, eukaryotic expression, purification, and identification of H-hFc fusion antibodies

[0850] Phage antibody clones NanoBCMA1.0, ..., NanoBCMA49 and NanoBCMA54, along with the positive control antibody BB2121, were redesigned and constructed into a eukaryotic expression vector containing IgG1 Fc. Primers were designed to amplify the phage antibody clone VHH by PCR. The PCR product was then recombinantly cloned into the pcDNA3.4-hFc vector, which was digested with SfiI and NotI restriction endonucleases. After confirming correct Sanger sequencing, the plasmid was transfected into Expi293 cells for transient expression. The protein was purified using a Protein A affinity column. The BCMA VHH-hFc proteins of the antibody and the positive control antibody were subjected to HPLC-SEC and SDS-PAGE for monomer purity identification, as shown in Figures 4 and 5 and Table 3. The overall monomer purity of the purified proteins was high, which can be used for subsequent protein-level functional identification.

[0851] Table 3. Monomer purity ratio of BCMA-targeting nanobodies by HPLC-SEC and SDS-PAGE

[0852] Example 3. Detection of BCMA-targeting nanobody binding activity to BCMA protein using ELISA

[0853] The binding activity of BCMA VHH-hFc prepared in Example 2 to BCMA protein was determined using an ELISA assay. In short, human BCMA was... ECD- His protein was diluted to a final concentration of 1 μg / mL, 100 μL / well, and coated onto a 96-well ELISA plate. The plate was then incubated overnight at 4°C. Wash the ELISA plate three times with PBST (0.05% TWEEN-20), and block the ELISA plate with 5% skim milk powder at room temperature for 1 hour. After blocking, wash the ELISA plate three times with PBST, and add serially diluted NanoBCMA1-hFc, NanoBCMA3.2-hFc, ..., NanoBCMA53-hFc and NanoBCMA54-hFc, positive controls hBCMA22-hFc and BB21221-hFc, and incubate at room temperature for 2 hours. After washing the ELISA plate three times with PBST, add Goat Anti-human IgG Fc-HRP (1:5000) and incubate at room temperature for 1 hour. After washing the ELISA plate seven times with PBST, add TMB for color development, incubate at room temperature in the dark for 5 minutes, and terminate the reaction with ELISA stop solution. Measure the OD value of each well at 450 nm using a microplate reader.

[0854] As shown in Figure 6 and Table 4, the exemplary BCMA-targeting nanobodies exhibit varying degrees of binding activity with the BCMA protein. 50 The numerical range was 0.03071-0.2149 μg / mL, OD 450 The highest values ​​range from 2.376 to 3.233, EC 50 The values ​​were all lower than the positive control BB2121-hFc (0.2274 μg / mL), among which NanoBCMA1.0-hFc (OD 450 =3.233), NanoBCMA29-hFc(OD 450 =2.844), NanoBCMA37-hFc(OD 450 =2.820) The highest binding signal thresholds were all higher than those of the positive control BB2121-hFc (OD) 450 =2.666), indicating that the BCMA antibody prepared in this application has high binding activity to BCMA protein.

[0855] Table 4. Results of the binding activity of anti-BCMA antibody to BCMA protein.

[0856] Example 4. Detection of BCMA receptor expression level in human myeloma cell lines

[0857] The expression levels of BCMA antigen on the surface of K562, MOLP8, MM.1S and NCIH929 cells were detected by quantitative flow cytometry. The positive rate and molecular weight of BCMA expression in target cells were detected by BD flow cytometer using PE anti-human BCMA (570546, BD Biosciences) antibody staining and flow cytometry quantitative kit (340495, BD Biosciences).

[0858] As shown in Figure 7, the negative control cell K562 basically does not express BCMA target antigen, while wild-type human myeloma cell lines MOLP8, MM.1S and NCIH929 cells specifically express BCMA target antigen, and the expression efficiency is all above 80%. Among them, the abundance of BCMA antigen expression on the surface of NCIH929 cells is higher than that of MOLP8 and MM.1S, with the number of antigen molecules reaching as high as 365,400 per cell.

[0859] Example 5. Detection of the binding activity of BCMA-targeting nanobodies to target cells with different BCMA antigen expression abundances.

[0860] Based on flow cytometry fluorescence sorting (FACS), an iQue Screener flow cytometer (purchased from IntelliCyt) was used to measure NanoBCMA1-hFc, ..., NanoBCMA53-hFc, NanoBCMA54-hFc, hBCMA22-hFc, and B38MV. H H1-hFc, B38MV H The binding activities of H2-hFc and LCARB38M-hFc with target cell lines CHO-hBCMA, NCIH929, and MOLP8 with different BCMA target antigen expression levels were investigated. In short, PBS containing 0.1% BSA was used as a buffer to prepare a final concentration of 1x10⁻⁶. 6 Target cells (CHO-hBCMA, NCIH929, MOLP8) were added to 96-well conical plates (corning 3894), 30 μL per well. 100 nM antibody was prepared and serially diluted 2.5 or 3-fold to create 11 concentration gradients. 30 μL of each antibody concentration was added to the plated target cells and mixed thoroughly. The cells were incubated at 4°C for 1 h. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 min, and the supernatant was discarded. The cells were then gently shaken to loosen the cells. The washing was repeated once. 30 μL of flow cytometry secondary antibody (ab98593) was prepared at a 1:200 ratio using buffer and added to each well. The cells were then incubated at 4°C for 30 min. 150 μL of buffer was added to each well, centrifuged at 300 g for 5 min, and the supernatant was discarded. The cells were then gently shaken to loosen the cells. The washing was repeated once. 35 μL of buffer was added to each well and mixed thoroughly before flow cytometry analysis.

[0861] As shown in Figures 8-10 and Table 5, on CHO-hBCMA target cells with high BCMA antigen expression abundance, EC 50 The numerical range was 0.3389–9.73 μg / mL; on NCIH929 target cells with relatively high BCMA antigen expression abundance, EC 50 The numerical range was 0.10–7.30 μg / mL; on MOLP8 target cells with relatively low BCMA antigen expression abundance, EC 50 The values ​​ranged from 0.04892 to 2.18 μg / mL, and the negative control PBS showed no binding activity. In summary, the exemplary BCMA-targeting nanobody exhibits binding activity and affinity with target cell lines expressing different BCMA target antigens.

[0862] Table 5. ECs targeting BCMA nanobodies binding to different cell surface BCMA receptors 50 Value (μg / mL)

[0863] Example 6. Detection of surface plasmon resonance (SPR) affinity of BCMA-targeted nanobodies

[0864] Based on biomembrane interferometry (BLI) technology, the binding kinetics of antigen and antibody were analyzed using a Fortebiooctet RED384 molecular interaction analyzer. The binding kinetics of NanoBCMA1-hFc, NanoBCMA3.2-hFc, ..., NanoBCM49-hFc, and NanoBCMA54-hFc to BCMA were measured. ECD - Affinity to His proteins. In short, using PBS (FB buffer) containing 0.1% BSA and 0.02% TWEEN-20 as a buffer, BCMA... ECD - Dilute His protein to 50 nM and add 200 μL / well to a 96-well microplate; add serially diluted NanoBCMA1-hFc, NanoBCMA3.2-hFc, ..., NanoBCMA49-hFc and NanoBCMA54-hFc samples, 200 μL / well; immobilize BCMA using a HISK sensor. ECD -His protein was bound for 3 min; then bound to a 2-fold diluted antibody solution for 3 min, followed by dissociation for 5 min. The obtained data were analyzed using Octet Data Analysis 10.0 software (Fortebio) (Langmuir, local fitting, 1:1 binding model) to calculate the antibody-antigen affinity KD value, binding constant Ka (1 / Ms) value, and dissociation constant Kd (1 / s) value.

[0865] As shown in Figure 11 and Table 6, the regression equation for the detection of exemplary BCMA-targeting nanobodies exhibits a goodness-of-fit R² value greater than 95% for the sample data points, indicating a reliable detection model. The exemplary BCMA-targeting nanobodies possess varying levels of affinity. The affinity of NanoBCMA9.2-hFc, NanoBCMA29-hFc, and NanoBCMA37-hFc ranges from 3.28E-10M to 6.72E-11M, while the affinity of the remaining BCMA-targeting nanobodies is on the order of -10. Overall, the BCMA-targeting nanobodies exhibit high sequence affinity.

[0866] Table 6. Affinity between BCMA-targeting nanobodies and human BCMA protein

[0867] Ka: Binding rate constant; Kd: Dissociation rate constant; KD: Affinity constant.

[0868] Example 7. Epitope mapping and epitope difference identification of BCMA-targeting nanobodies

[0869] 7.1 Epitope peptide binding activity

[0870] Human BCMA (UniPro identifier: Q02223-1) is a single-pass transmembrane protein of 184 amino acids in length. As shown in Table 7, human BCMA consists of an extracellular domain (ECD, amino residues 1-54), a transmembrane domain (TM, amino residues 55-77), and a cytoplasmic domain (CD, amino residues 78-184). The UniProt database (https: / / www.uniprot.org / uniprotkb / Q02223 / entry) shows that three disulfide bonds (Cys-Cys) are formed at positions 8-21, 24-37, and 28-41 on the ECD of BCMA. The secondary structure of BCMA ECD, from the N-terminus to the C-terminus, consists of β chains (AA12-15), turns (AA16-19), β chains (AA20-23), helices (AA24-27), β chains (AA30-32), helices (AA35-37), turns (AA38-40), and turns (AA42-44). The crystal structure of human BCMA ECD is shown in Figure 12.

[0871] Table 7. Amino acid sequence and position of human BCMA

[0872] Table 8. Human BCMA ECD Linearized epitope polypeptide sequence

[0873] According to the BCMA diagramECD The crystal structure and amino acid sequence and position of human BCMA are shown in Table 7 and Table 8. Linear BCMA epitope peptides (BCEP001-BCEP007) were designed and synthesized by Genscript Biotech Co., Ltd., followed by N-terminal biotinylation. The binding activity of NanoBCMA1-hFc, NanoBCMA3.2-hFc, ..., NanoBCMA49-hFc and NanoBCMA54-hFc to the linearized BCMA epitope peptides was determined using an ELISA assay. In summary, the synthesized BCEP001-BCEP007 epitope peptides were diluted to a final concentration of 1 μg / mL with PBS, and 200 μL / well was coated onto 96-well ELISA plates and incubated overnight at 4°C. Wash the plate three times with PBST (0.05% TWEEN-20), and block the ELISA plate with 0.5% BSA at room temperature for 2 hours. After blocking, wash the ELISA plate three times with PBST, and add 100 nM final concentrations of NanoBCMA1-hFc, NanoBCMA3.2-hFc, ..., NanoBCMA49-hFc and NanoBCMA54-hFc to two copies, and incubate at room temperature for 2 hours. After washing the ELISA plate three times with PBST, add 100 μL of Goat Anti-human IgG Fc-HRP (1:8000) to each well and incubate at room temperature for 1 hour. After washing the ELISA plate seven times with PBST, add TMB for color development, and incubate at room temperature in the dark for 5-10 minutes. Terminate the reaction with ELISA stop solution, and measure the absorbance of each well at 450 nm using a microplate reader.

[0874] As shown in Figure 13, among the exemplary BCMA-targeting nanobodies, NanoBCMA1-hFc, NanoBCMA3.2-hFc, and NanoBCMA11-hFc exhibit the strongest binding activity to the BCEP002 peptide (amino acids 8-21 of BCMA ECD), mainly containing secondary structures of β-chain (AA12-15), turn (AA16-19), and β-chain (AA20-23); NanoBCMA29-hFc exhibits the strongest binding activity to the BCEP005 peptide (amino acids 24-42 of BCMA ECD), mainly containing secondary structures of helix (AA24-27), β-chain (AA30-32), helix (AA35-37), and turn (AA38-40); NanoBCMA32-hFc, NanoBCMA37-hFc, and NanoBCMA40-hFc exhibit binding activity to multiple linearized epitopes. In summary, different exemplary BCMA-targeting nanobodies exhibit different binding epitopes. Combining BCMA nanobodies with different or partially overlapping epitopes can produce dual or multi-epitope binding antibodies, which can generate synergistic or enhanced efficacy.

[0875] 7.2 SPR competitive binding activity

[0876] Based on biomembrane interference (BLI) technology, the kinetics of epitope differences between exemplary BCMA-targeting nanobodies were analyzed using a Fortebiooctet RED384 molecular interaction analyzer. Briefly, NanoBCMA1-hFc, NanoBCMA3.2-hFc, NanoBCMA9.2-hFc, and NanoBCMA11-hFc were prepared to a final concentration of 100 nM using PBS (FB buffer) containing 0.1% BSA and 0.02% TWEEN-20, and added to 96-well plates at 200 μL / well; BCMA was then added to a final concentration of 50 nM. ECD -His protein, 200 μL / well; BCMA immobilized using a HISK sensor. ECD The HISK sensor immobilized the BCMA protein, which was then saturated with NanoBCMA9.2-hFc, hBCMA22-hFc, NanoBCMA1-hFc, NanoBCMA3.2-hFc, and NanoBCMA11-hFc, respectively. Using NanoBCMA9.2-hFc as an example, the HISK sensor immobilized BCMA. ECD After binding to the His protein, NanoBCMA9.2-hFc was pre-saturated. After washing, it was re-bound with NanoBCMA1-hFc, NanoBCMA3.2-hFc, and NanoBCMA11-hFc, respectively. The presence of affinity binding activity of NanoBCMA1-hFc, NanoBCMA3.2-hFc, and NanoBCMA11-hFc after saturation binding with NanoBCMA9.2-hFc was evaluated. As a control, NanoBCMA9.2-hFc was also bound simultaneously to assess complete saturation binding. Similarly, saturation binding identification of other exemplary BCMA-targeting nanobodies was performed. The obtained data were analyzed using Octet Data Analysis 10.0 software (Fortebio) to calculate the antibody-antigen binding strength, affinity KD value, binding constant Ka (1 / Ms), and dissociation constant Kd (1 / s).

[0877] As shown in Figure 14, after saturation binding of NanoBCMA9.2-hFc, hBCMA22-hFc maintained its binding activity, with a response value of 0.3427. This may be because the binding epitope of hBCMA22-hFc is different from that of NanoBCMA9.2 antigen, and the binding does not interfere with each other; or it may be that the epitope is the same, but hBCMA22 has higher affinity and competes for binding to BCMA. ECD-His protein; other exemplary nanobodies do not exhibit affinity binding activity. As shown in Figure 15, after saturation binding of hBCMA22-hFc, NanoBCMA9.2-hFc shows no affinity binding activity, with a response value of 0.0244. This may be due to the same epitope as the NanoBCMA9.2 antigen-binding epitope, or spatial conflict and overlap of epitopes. As shown in Table 9, BCMA ECD - After the His protein saturates and binds to NanoBCMA26-hFc and NanoBCMA28-hFc, LCARB38M exhibits binding activity with response values ​​of 0.1167 and 0.2143, respectively. This suggests that NanoBCMA26, NanoBCMA28, and LCARB38M have different antigen-binding epitopes and exhibit epitope differences. Combining BCMA nanobodies with different or partially overlapping epitopes can produce dual or multi-epitope binding antibodies, which can generate synergistic or enhanced efficacy.

[0878] Table 9. Identification of differential SPR epitopes of BCMA-targeting nanobodies

[0879] 7.3 FCM competitive binding activity

[0880] Competitive binding assays using flow cytometry were used to verify the differential epitopes between different BCMA-targeting nanobodies. In summary, exemplary BCMA-targeting nanobodies were biotinylated using a biotinylation kit (ab201795, Abcam), and the labeling efficiency was verified by Fortebio. As shown in Figure 16 and Table 10, NanoBCMA1-hFc, NanoBCMA9.2-hFc, and NanoBCMA37-hFc were successfully biotinylated, and EC50 binding between the BCMA-targeting nanobodies and target cells was obtained. 50 and EC 80 The numerical value (μg / mL) corresponds to EC 80 Antibodies at a given concentration are defined as saturating antibodies, and antibodies that are used to identify epitope differences are defined as competitive antibodies.

[0881] According to EC 80 The corresponding saturation antibody concentration and 3x10 4CHO-hBCMA or NCIH929 target cells were incubated for 1 hour. A competitive antibody of 375 nM was prepared using PBS buffer and serially diluted 5-fold to create eight concentration gradients (final concentration 0). The serially diluted competitive recombinant antibody was then added to each well and incubated at 4°C for another 1 hour. After incubation, 140 μL of buffer was added to each well, centrifuged at 300 g for 5 min, and the supernatant was discarded. The cells were washed once. Based on the antibody tag, APC-streptavidin or PE-anti-mouse Fc fluorescent secondary antibody was prepared at a 1:200 ratio using buffer. 30 μL of this antibody was added to each well and mixed, then incubated at 4°C for 30 min. 170 μL of buffer was added to each well, centrifuged at 300 g for 5 min, and the supernatant was discarded. The cells were washed once. 35 μL of buffer was added to each well and mixed before analysis using an iQue Screener flow cytometer. As a control, following the same procedure described above, serially diluted competitive antibodies were directly incubated with CHO-hBCMA or NCIH929 target cells in the absence of saturating antibodies to detect the binding activity of the competitive antibodies to CHO-hBCMA or NCIH929 target cells in the absence of saturating antibodies.

[0882] As shown in Figure 17, after co-incubating Biotin-NanoBCMA29 with different candidate antibodies, the binding activity of Biotin-NanoBCMA29 and CHO-hBCMA in the co-incubation system was detected by APC-streptavidin. It can be seen that the exemplary BCMA-targeting nanobodies that did not completely compete for the binding of Biotin-NanoBCMA29 to CHO-hBCMA cells include NanoBCMA1, NanoBCMA3.2, NanoBCMA11, and NanoBCMA37, indicating that these antibodies may have different antigen-binding epitopes with NanoBCMA29, and their binding does not interfere with each other. It can also be seen that the exemplary BCMA-targeting nanobodies that completely compete for the binding of Biotin-NanoBCMA29 to CHO-hBCMA cells include NanoBCMA9.2, indicating that this antibody may have the same antigen-binding epitope as NanoBCMA29 and have higher affinity or a larger antigenic epitope than NanoBCMA29, indicating epitope overlap and conflict. As shown in Figure 18, NanoBCMA9.2 competitively binds to the other candidate antibodies. Among them, NanoBCMA1, NanoBCMA3.2, NanoBCMA11, NanoBCMA29, and NanoBCMA37 failed to fully compete for the binding of NanoBCMA9.2 to CHO-hBCMA cells, suggesting that they may have different binding epitopes than NanoBCMA29.

[0883] Table 10. EC50 and EC80 values ​​(μg / mL) of BCMA-targeting nanobodies binding to target cells.

[0884] In summary, the exemplary BCMA-targeting nanobody targets the BCMA target antigen and has different antigen recognition epitopes. The inventors' research shows that by simultaneously binding to different epitopes of the same antigen, the sensitivity and binding ability to specific target cells can be significantly enhanced, tumors can be cleared more efficiently, the clinical treatment effect of hematologic malignancies can be improved, side effects can be reduced, and safety can be guaranteed.

[0885] Example 8. Detection of the polymerization structure of BCMA-targeting nanobodies under antigen induction

[0886] Size exclusion chromatography (SEC) was used with high performance liquid chromatography (HPLC) to determine NanoBCMA1-hFc, NanoBCMA3.2-hFc, ..., NanoBCMA49-hFc and NanoBCMA54-hFc in BCMA. ECD -The polymerized structure formed under the induction of His protein. In short, through preliminary experiments, an antibody-to-antigen molar molecular weight ratio of 1:9 was selected. Based on the theoretical molecular weights of the antigen and antibody, the corresponding concentration of BCMA was prepared using 1xPBS buffer. ECD -His protein and NanoBCMA1-hFc, NanoBCMA3.2-hFc, ..., NanoBCMA49-hFc, NanoBCMA54-hFc; BCMA ECD The His protein and nanobody were mixed thoroughly in a 1.5 mL EP tube to form a complex; the mixture was shaken at 300 rpm for 2 min and then incubated at 37 °C for 60 min; the tube was then flushed with pure water at a flow rate of 2 mL / min for 30 min using a high-performance liquid chromatograph, the knob was tightened, and the tube was flushed at a flow rate of 0.5 mL / min for 30 min; the tube was then flushed with PBS at a flow rate of 1 mL / min for 1 h to equilibrate; 100 μL of the antigen-antibody complex after co-incubation was added to the HPLC built-in tube (avoiding air bubbles), the injection volume was set to 20 μL, and the sample was loaded for analysis.

[0887] Figure 19 illustrates exemplary BCMA-targeting nanobodies NanoBCMA1.0, NanoBCMA29, and BCMA. ECD -His binds in a monomeric form, and due to the bivalent structure of hFc, it can bind to two BCMAs simultaneously. ECD -His molecule; NanoBCMA9.2 and BCMA ECD -His binding occurs in a larger form than monomeric forms. In summary, different exemplary BCMA-targeting nanobodies and BCMA...ECD -His target proteins bind in different stoichiometric ratios. According to relevant research reports, the potential for polymerization of nanobodies under antigen induction is positively correlated with their expected therapeutic effects.

[0888] Example 9. Specificity evaluation of BCMA-targeting nanobodies

[0889] Based on flow cytometry fluorescence sorting (FACS) technology, exemplary BCMA V was measured using an iQue Screener flow cytometer (purchased from IntelliCyt). H The binding activity of H antibody to BCMA antigen-negative cell lines was investigated. In short, PBS containing 0.1% BSA was used as a buffer to prepare a final concentration of 1x10⁻⁶. 6 Target cell lines (in this example, normal or tumor cells that do not express BCMA antigen, such as human gastric epithelial cell line GES-1, human microvascular endothelial cell line HMEC-1, human pancreatic ductal epithelial cell line HPDE6-C7, human fibroblast cell line HS-27A, human ovarian surface epithelial cell line IOSE-80, human prostate cancer cell line PC-3, human urinary tract epithelial cell line SV-HUC-1, etc.) were added to 96-well conical plates. 3894), 30 μL per well; prepare detection antibody at a concentration of 200 nM, and serially dilute 5-fold to form 11 concentration gradients; add 30 μL of antibody at different concentrations per well to the plated target cells and mix well; incubate at 4°C for 1 h; add 150 μL of buffer to each well, centrifuge at 300 g for 5 min, discard the supernatant and loosen the cells by shaking; repeat washing once; prepare fluorescent secondary antibody (ab98593) at a 1:200 ratio using buffer, add 30 μL to each well to the cells and mix well, incubate at 4°C for 30 min; add 150 μL of buffer to each well, centrifuge at 300 g for 5 min, discard the supernatant and loosen the cells by shaking; repeat washing once; add 35 μL of buffer to each well, mix well, and then detect using flow cytometry.

[0890] As shown in Figure 20, the exemplary BCMA-targeting nanobody prepared in this application has virtually no non-specific binding with non-specific target cells, indicating that the antibody sequence has high specificity and a low off-target possibility, and can be used for in vivo and in vitro biological function verification at the CAR-T cell level.

[0891] Example 10. Construction of BCMA-specific chimeric antigen receptor lentiviral expression vector

[0892] The CAR lentiviral core empty vector plasmid (self-constructed by Yuanqi Biotechnology, containing the Ori2 novel element, hereinafter referred to as the CAR lentiviral empty vector) was double-digested with SphI (Cat.#R3182L, NEB) and NotI (Cat.#R3189L, NEB) restriction endonucleases to generate linearized fragments. After gel recovery, the fragments were mixed with the VHH fragments of NanoBCMA1.0, NanoBCMA3.2, NanoBCMA9.2, NanoBCMA11, NanoBCMA29, NanoBCMA32, NanoBCMA37, NanoBCMA49 and NanoBCMA54 obtained in Example 1 at a molar ratio of 1:3 for homologous recombination. The mixture was then transformed into DH5α competent cells, plated on kanamycin-resistant plates, and incubated overnight in an inverted CO2 incubator at 37°C. Single colonies were then picked and sequenced for identification. Figure 21 shows the components and their connection order in CAR lentiviral empty vectors and BCMA-specific CAR-Ori2 vectors (named NanoBCMA1.0-Ori2, NanoBCMA3.2-Ori2, NanoBCMA9.2-Ori2, NanoBCMA11-Ori2, NanoBCMA29-Ori2, NanoBCMA32-Ori2, NanoBCMA37-Ori2, NanoBCMA49-Ori2 and NanoBCMA54-Ori2, or named NanoBCMA1.0, NanoBCMA3.2, NanoBCMA9.2, NanoBCMA11, NanoBCMA29, NanoBCMA32, NanoBCMA37, NanoBCMA49 and NanoBCMA54).

[0893] Example 11. Packaging and titer determination of BCMA-specific chimeric antigen receptor lentivirus

[0894] The lentiviral vector system used to construct this invention is a third-generation system, consisting of four plasmids, as shown in Figure 22: the PRH1 plasmid encoding the Gag-Pol protein, the PMH2 plasmid encoding the envelope protein VSV-G, the PVH3 plasmid encoding the Rev protein, and a CD38 VHH core plasmid. The expression of the CAR gene in each CAR lentiviral plasmid is regulated by the elongation factor-1α (EF-1α) promoter. The lentiviral packaging process is as follows: 1x10 6293T cells were suspended in 2 mL of DMEM medium containing 10% FBS and cultured overnight in 6-well plates. 345 μL of the original medium was aspirated, and 345 μL of Opti-MEM medium (50 times the plasmid mass) containing 6.90 μg of packaging plasmid (PRH1:PMH2:PVH3:core plasmid = 2:1:1:2) and 20.7 μL of FuGENEHD transfection reagent (Cat.#E2311, Promega) was added evenly. The mixture was gently mixed and incubated at 37°C for 12 hours in a CO2 incubator. The plasmid-containing medium was removed, and the cells were washed once with PBS and replaced with 2 mL of DMEM medium containing 5% FBS. The cells were cultured for 48 hours. 2 mL of viral supernatant was collected, centrifuged at 3000 rpm for 10 min, aliquoted, and stored at -80°C for later use. The viral titer was also determined.

[0895] When the positive rate of each test tube minus the negative control is 5%-20%, the results are usable, i.e.:

[0896] The titers of the aforementioned CAR viruses containing VHH ranged from 5 to 10 × 10⁻⁶. 6 TU / mL.

[0897] Example 12. Preparation of CAR-T cells and lentiviral infection

[0898] In this embodiment, CD3 microbeads reagent kit (Miltenyi Biotec, Cat. #130-097-043) was used to sort CD3 in PBMCs. + T cells, specifically, were revived using PBMCs (Ficoll density gradient centrifugation), centrifuged at 500g for 5 minutes to remove cryopreservation medium. They were then treated with 1×10⁻⁶ cells. 7 Resuspend the cells in 80 μL of pre-cooled sorting buffer. Repeat at a rate of 1 × 10⁶ cells / mL. 7 Add 20 μL of LCD3 magnetic beads to the total cell count and mix thoroughly. Incubate at 4°C for 15 min. After incubation, repeat the process at 1×10⁻⁶ cells / mL. 7 Add 1.5 mL of pre-cooled sorting buffer to the total cell count, centrifuge at 500 g for 5 min, and discard the supernatant. Repeat at 1 × 10⁻⁶. 7 Resuspend the cells in 0.1 mL of room temperature separation buffer, mix thoroughly by pipetting, and transfer the cell suspension to a rinsed LS separation column (placed under a magnetic field). Collect CD3+ T cells, mix thoroughly, centrifuge at 500 g for 5 min, discard the supernatant, and adjust the cell density to 1.25 × 10⁻⁶ cells in X-VIVO complete medium. 6 T cells were activated by adding CD3 / CD28 DynaBeads magnetic beads at a cell number:bead number ratio of 1:3. The cells were cultured at 37°C and 5% CO2. PBMCs were then sorted using CD3-positive magnetic beads to obtain CD3-positive DynaBeads with a purity >95%.+ T cells.

[0899] This will activate CD3 for 24 hours. + T lymphocytes were collected in 50 mL centrifuge tubes, centrifuged at 500 g for 5 min, the culture supernatant was discarded, and the cells were resuspended in X-VIVO complete medium for counting. The cells were counted at a rate of 7 × 10⁻⁶ cells / mL. 5 The total cell count was determined, and cells were seeded into 12-well plates with a multiplicity of infection (MOI) of 3. The required volume of lentivirus was added, and polybrene reagent was added to a final concentration of 10 μg / mL. The mixture was thoroughly mixed, centrifuged at 1200 rpm for 60 min, and cultured at 37°C with 5% CO2. After 24 h, cells were collected, centrifuged at 500 g for 5 min to remove the supernatant, and resuspended in X-VIVO complete medium to adjust the cell density to 5-7 × 10⁶ cells / well. 5 The cells were inoculated at a concentration of 10 cells / mL into 12-well cell culture plates and cultured at 37°C with 5% CO2.

[0900] Example 13. Detection of CAR gene expression efficiency and in vitro amplification

[0901] 13.1 CAR gene expression detection

[0902] CD3 / CD28 DynaBeads-activated T cells, after lentiviral infection and 72 hours of culture, were used to detect the T cell CAR gene expression efficiency, as follows:

[0903] (1) Mix the cells, count the AO / PI ratio, and detect the CAR-T cell density. Take 4×10⁶ cells. 5 The total number of cells was transferred to a flow cytometry tube, 2 mL of 1xPBS (containing 2% FBS) buffer was added, the mixture was vortexed and centrifuged at 1600 rpm for 5 min.

[0904] (2) Remove the supernatant and resuspend the cells in 50 μL of 1xPBS (containing 2% FBS) buffer.

[0905] (3) Add 1 μL of APC-labeled BCMA-His antibody, mix thoroughly, and incubate at 4°C for 30 min.

[0906] (4) Add 1 mL of 1xPBS (containing 2% FBS) buffer to wash, vortex to mix, and centrifuge at 1600 rpm for 5 min.

[0907] (5) Wash once more.

[0908] (6) Resuspend in 300 μL of 1xPBS (containing 2% FBS) buffer, mix thoroughly, and then perform detection.

[0909] As shown in Figures 23 and 24, after activation and amplification, the CAR positivity rate of T cells 72 hours after BCMA-specific CAR lentivirus infection ranged from 24% to 94%; and the CAR gene was stably expressed on T cells at different time points, with no significant differences among the groups.

[0910] 13.2 In vitro expansion of CAR-T cells

[0911] CAR-T cells activated by CD3 / CD28 DynaBeads were infected with lentivirus and passaged according to culture times Day 5, Day 7, Day 9, and Day 12. After thorough mixing of the CAR-T cells, 20 μL of the cell suspension was transferred to a 1.5 mL EP tube, and an equal volume of AO / PI reagent was added. 20 μL of the mixture was then added to a counting chamber, and cell counts were performed using an Auto2000 cell counter against a fluorescent background. Based on the obtained cell density, cell growth was recorded, including cell density, cell viability, and cell diameter, and a growth curve was plotted. Simultaneously, the cell passage density was adjusted to 7 × 10⁶ cells / year. 5 Cells / mL were seeded in 6-well plates and cultured for 10-12 days; data were processed and analyzed using GraphPad 10.0.

[0912] As shown in Figure 25, after in vitro CD3 / CD28 stimulation, the viability of CAR-T cells gradually increased with the extension of culture time, reaching 70-80% by Day 12. As shown in Figure 26, the diameter of CAR-T cells gradually decreased with the extension of culture time, indicating a tendency towards a resting state. The diameters of NanoBCMA1.0-Ori2, NanoBCMA3.2-Ori2, NanoBCMA11-Ori2, and NanoBCMA29-Ori2 cells were close to those of the Mock T control cells by Day 12. As shown in Figure 27, after in vitro CD3 / CD28 stimulation, CAR-T cells proliferated stably and can be used for CAR-T cell functional experiments.

[0913] Example 14. Evaluation of the in vitro targeted killing activity of BCMA CAR-T cells

[0914] CAR-T cells activated by CD3 / CD28 DynaBeads were cultured for 9 days, and their effector killing activity was assessed using the Luciferase Assay System kit (Cat.#E6120, Promega). In summary, the total number of target cells expressing the Luciferase gene was 3.3 x 10⁻⁶. 4Cells were seeded per well in a 96-well plate. The prepared CAR-T cells were pre-tested for positivity and mixed with target cells at different ratios (E:T) of 3:1, 1:1, and 1:3, with three replicates per group. The cells were co-cultured at 37°C in a 5% CO2 incubator for 24 hours. After co-culture, the cells were thoroughly mixed, and 100 μL of the cell suspension was transferred to an opaque 96-well plate. 100 μL of Luciferase Assay Substrate was added, mixed thoroughly, and incubated at room temperature in the dark for 5 minutes. The luciferase luminescence intensity in the remaining target cells was measured using a multi-functional microplate reader. The killing efficiency was quantitatively assessed using the luciferase in the remaining tumor cells after killing.

[0915] Formula for calculating cell kill ratio:

[0916] As shown in Figure 28, compared with blank Mock T cells, the results showed that the different BCMA-specific CAR-T cells prepared in this application had significant killing activity against MOLP8 target cells, and this activity was gradient-dependent. Among them, at low-efficiency target ratios of 1:3 and 1:9, the exemplary NanoBCMA3.2, NanoBCMA29, and NanoBCMA35 CAR-T cells showed significantly higher killing rates against MOLP8 target cells than the positive control antibody B38MV. H H1, B38MV H H2 (patent CN109153731B).

[0917] Example 15. Assessment of BCMA CAR-T cytokine secretion levels

[0918] Exemplary BCMA-targeted CAR-T cells were cultured in vitro for 9 days, and the levels of CAR-T cytokines IL-2 and IFN-γ were detected using an IL-2 and IFN-γ cytokine ELISA kit from R&D Corporation. The positivity rate and cell density of CAR-T cells were pre-tested. Lentivirally infected CAR-T cells were adjusted with blank T cells to ensure consistent cell counts across groups. CAR-T cells and MM.1S target cells were co-incubated at an effector-to-target ratio of 1:1, with a total CAR-positive cell count of 3 × 10⁻⁶ cells. 4 The total number of MM.1S cells was 3 × 10⁻⁶. 4Each group was set up with 3 replicates. After incubation for 24 hours, the plates were centrifuged at 250g for 5 minutes, and the supernatant was collected. The cytokine secretion levels were detected using the corresponding IL-2 and IFN-γ ELISA kits. In short, the captured antibody was coated onto a 96-well ELISA plate at a concentration of 1 μg / mL and incubated overnight at 4°C. Non-specific binding sites were blocked with 5% skim milk powder. After thorough washing, the supernatant of the sample, diluted 3 times, was added to the 96-well plate and incubated at room temperature for 2 hours. After thorough washing, biotinylated detection antibody working solution (100 μL / well) was added and incubated at room temperature for 2 hours. After thorough washing, enzyme conjugate working solution (100 μL / well) was added and incubated at room temperature in the dark for 20 minutes. After thorough washing, TMB chromogenic reagent (100 μL / well) was added and incubated at room temperature in the dark for 20 minutes. Finally, ELISA stop solution (50 μL / well) was added, and the OD value of each well was measured at 450 nm.

[0919] As shown in Figures 29 and 30, under MOLP8 target cell stimulation, the IL-2 and IFN-γ secretion levels of the different BCMA-specific CAR-T cells (NanoBCMA3.2, NanoBCMA9.2, NanoBCMA29, and NanoBCMA37) prepared in this application were all higher than those of the Mock T cell group; and compared with the positive control antibody B38MV, the IL-2 and IFN-γ secretion levels were also higher. H H1 and B38MV H In H2, the IFN-γ secretion level of the antibody in this application was significantly enhanced, indicating a strong antigen-specific activation effect of CAR-T cells.

[0920] Example 16. Assessment of BCMA CAR-T cell targeted proliferation capacity

[0921] In this embodiment, untreated MM.1S cells were used as target cells for the detection of BCMA-specific CAR-T cell targeting proliferation. CAR-T cells activated by CD3 / CD28 DynaBeads and control Mock T cells were infected with lentivirus, and their targeting proliferation capacity was detected according to culture time Day 9 or Day 12. The CAR positivity rate of each group was detected in advance, and then the CAR positivity rate of each group was adjusted to be consistent using blank effector T cells (the culture medium was X-VIVO without any additives). 15(Blank culture medium). CAR-T cells and MM.1S cells were subjected to the first round of targeted stimulation at an effector-to-target ratio of 1:1. After co-incubation for 4-5 days, all cells were collected, and cell counts were performed using AO / PI reagents. Cell density and cell diameter were measured. Then, a second round of targeted stimulation was performed at a ratio of 1:3 (CAR-T cells:MM.1S cells). After another 4-5 days of co-incubation, a third round of targeted stimulation was performed (the ratio of the two cell types was the same as in the second round). During the three rounds of targeted stimulation, an appropriate amount of X-VIVO was added every 1-2 days according to cell growth. 15 Blank culture medium. Count the number of CAR-T cells in each round and plot the proliferation curve.

[0922] As shown in Figures 31-33, in the CAR-T and MM.1S target cell co-incubation system, all CAR-T cell groups were able to effectively kill tumor cells, while the Mock T cell control group still had a relatively high proportion of target cells remaining. In addition, under repeated stimulation by target cells, the CAR positivity rate of CAR-T cells in each group was upregulated and maintained, among which the NanoBCMA11 CAR positivity rate was relatively high.

[0923] Example 17. Study on BCMA CAR-T cell subtypes and phenotypes

[0924] This embodiment uses flow cytometry (FACS) to detect CAR-T cell phenotype, providing an important immune monitoring indicator for CAR-T cell therapy. In short, CD3+ activation after lentiviral infection... + After T cells achieved positive BCMACAR expression, their subtypes, memory phenotypes, activation phenotypes, and exhaustion phenotypes were detected on Day 9 of co-culture. Effector CAR-T cells were collected at the corresponding culture days, and appropriate cell counts were analyzed by flow cytometry for CD3 content in each group. + T cell subtype markers: including CD3-PB, CD4-APC, and CD8-Percpcy5.5; detection of CD3 in each group + Memory markers in T cells: including the proportion of CD4-APC, CD8-Percpcy5.5, CD45RA-BV510, CCR7-PE, and CD95-PECY7 cells, for activated T cells (CD3+). + CD95 + CD45RA and CCR7 can be used as the minimum marker combination to distinguish memory T cells at different stages, specifically classifying them into T cells. SCM (CD45RA + CCR7 + ), T CM (CD45RA - CCR7 +), T EM (CD45RA - CCR7 - ), T EFF (CD45RO + CCR7 - ); Detect CD3 in each group + Activating markers in T cells: including the proportion of CD3-BV510, CD137-PE, CD25-PE-CY7, and CD69-APC cells; detection of CD3 in each group. + Exhaustion markers in T cells: including the proportion of CD3-Percpcy5.5, PD-1-BV421, LAG-3-AF647, and TIM-3-BV510 cells.

[0925] As shown in Figures 34 and 35, the CD4 / CD8 ratios of BCMA-specifically targeted CAR-T cells from different groups were similar. At Day 9 of in vitro culture, the overall cell type was biased towards the CD4 subtype, with no significant differences between groups. As shown in Figures 36 and 37, at Day 9 of CAR-T cell culture, NanoBCMA29 CAR-T cells exhibited higher stem cell memory (T... SCM ) phenotype, lower central memory T cells (T CM The CAR-T cells exhibit a low degree of T cell differentiation. As shown in Figure 38, during Day 9 of CAR-T cell culture, the expression levels of the CAR-T cell activation markers CD137, CD25, and CD69 (NanoBCMA3.2, NanoBCMA11, and NanoBCMA29) were relatively low, indicating low T cell activation and a low-self-activation phenotype advantage. As shown in Figure 39, during Day 9 of CAR-T cell culture, the expression levels of the CAR-T cell exhaustion markers LAG-3 and TIM-3 (NanoBCMA29) were relatively low, indicating low T cell exhaustion and a low-exhaustion phenotype advantage.

[0926] Example 18. Construction of anti-BCMA bivalent CAR-T cell lentiviral expression vector

[0927] To obtain anti-BCMACAR-T cell products with stronger affinity and better anti-tumor effects, we combined six antibody sequences—NanoBCMA1.0, NanoBCMA3.2, NanoBCMA9.2, NanoBCMA11, NanoBCMA29, and NanoBCMA37—that exhibited good target cell killing activity, high targeted proliferation capacity, and low tonic signal levels in in vitro functional validation, with different peptide linkers to construct bivalent VHH CARs with BCMA binding domains. Exemplary bivalent BCMACAR constructs are shown in Table 11. The construct preparation steps are as follows: Specifically, two VHH sequences were fused using (GGGGS)n. Then, the CAR lentiviral core empty vector plasmid (same as in Example 10, self-constructed by Yuanqi Biotechnology, containing the Ori2 novel element, hereinafter referred to as the CAR lentiviral empty vector) was double-digested with SphI and NotI endonucleases (purchased from NEB) to generate a linearized fragment. After gel extraction and recovery, the fragment was mixed with the fused double VHH fragment at a molar ratio of 1:3 for homologous recombination and transformed into DH5α competent cells. Single colonies were picked and sequenced for identification. The connection sequence of the bivalent BCMACAR structure is detailed in Table 11.

[0928] Table 11. Exemplary Bivalent BCMACAR Structure Order

[0929] Example 19. Detection of CAR gene expression efficiency and in vitro amplification in anti-BCMA bivalent CAR-T cells

[0930] The specific experimental methods for detecting CAR gene expression efficiency and in vitro amplification are as described in Example 13. As shown in Figure 40, when the lentiviral infection multiplicity of infection (MOI) was 3–5, the CAR gene was stably expressed in anti-BCMA bivalent CAR-T cells from different PBMC-derived T cells, and the transduction efficiency in T cells ranged from 28.4% to 60.1%, which can be used for subsequent CAR-T cell function verification. As shown in Figure 41, the total expansion fold of anti-BCMA bivalent CAR-T cells after activation and culture for 12 or 14 days was between 100 and 570 times; as shown in Figure 42, the viability of anti-BCMA bivalent CAR-T cells after activation and culture for 12 or 14 days gradually increased with prolonged culture time, maintaining a cell viability of approximately 95%.

[0931] Example 20. Evaluation of in vitro effector killing activity of anti-BCMA bivalent CAR-T cells

[0932] CAR-T cells activated by CD3 / CD28 DynaBeads were infected with lentiviruses and subjected to CAR-T cell cytotoxicity experiments at day 9 of culture. MOLP8 cells overexpressing the Luciferase gene were used as target cells. Different effector-target ratios (1:1, 1:3, 1:9) were set, and free BCMA was added to simulate the in vivo microenvironment. The CAR-T cell killing efficiency was assessed, as detailed below:

[0933] (1) Count the target cells MOLP8-Luciferase-puro and, based on the counting results, take 3.3 × 10⁻⁶ cells. 4 Target cells were seeded into 96V plates at 100 μL per well, in duplicate. Blank X-VIVO buffer was used to prepare BCMA-His protein at a concentration of 3333 ng / mL (total required). Target cells were incubated with X-VIVO containing BCMA-His. A control group was also set up (target cells incubated with blank X-VIVO).

[0934] (2) Incubate at 37℃ for 1-2 hours;

[0935] (3) Based on the CAR-T cell count results and positive rate, take out the corresponding number of CAR-T cells and add the required amount of negative CAR-T cells for each group. Centrifuge at 4℃, 500g for 5min, resuspend with X-VIVO15 and plate, 100μL per well, 2 parallel wells.

[0936] (4) The remaining CAR-T cells were serially diluted 3-fold. The diluted CAR-T cells were seeded into the corresponding wells containing target cells at an effector-to-target ratio of 1:3, 100 μL per well, in parallel with two wells.

[0937] (5) Repeat step (5) to lay the corresponding orifice plate with a target ratio of 1:9;

[0938] (6)(7) Incubate at 37℃ and 5% CO2 for 20-24 hours;

[0939] (7) After 24 hours, remove the 96-well plate with the bottom of the v plate, mix the cells, take Steady-Glo out of the -20℃ freezer, transfer 50 μL of the mixed cells to each well of the opaque 96-well plate, and then add 50 μL of Steady-Glo fluorescent agent to each well, taking care to avoid generating bubbles. Incubate for 5 minutes and then read the results with a microplate reader.

[0940] As shown in Figures 43 and 44, in the validation of T cells derived from different PBMCs, compared with blank effector T cells, the results showed that anti-BCMA bivalent CAR-T cells could specifically kill target cells MOLP8-Luci-puro. In blank culture medium, anti-BCMA bivalent CAR-T cells showed better killing effect, with a killing efficiency of 40%–60% at an effector-to-target ratio of 1:3. After the addition of free BCMA, although the BCMA sequence function of anti-BCMA bivalent CAR-T cells was partially blocked, they still retained killing activity.

[0941] Example 21. Detection of anti-BCMA bivalent CAR-T cytokine secretion

[0942] To evaluate the level of IFN-γ cytokine secreted by anti-BCMA bivalent CAR-T cells after co-incubation with target cells under conditions of free BCMA and without free BCMA. Anti-BCMA bivalent CAR-T cells cultured to Day 9 were co-incubated with the BCMA-low-expressing cell line MOLP8 at effector-to-target ratios of 1:1, 1:3, and 1:9, with the MOLP8 cell number fixed at 3.3 × 10⁻⁶ cells / year. 4 Cells were placed per well. Based on the CAR-T cell positivity rate, blank T cells were used to adjust the CAR-T positivity rate of each group to be consistent. Target cells in the group containing free BCMA were pre-incubated with a certain amount of BCMA-his protein for 1-2 hours, with the remaining procedures being the same. The cells were plated according to the experimental settings and co-incubated at 37℃ for 24 hours. After centrifugation at 250g for 5 minutes, the supernatant was collected, and the cytokine secretion level was detected using an IL-2 and IFN-γ cytokine ELISA kit from R&D Company. As shown in Figure 45, under conditions with and without free BCMA, different combinations of bivalent BCMA CAR-T cells, after co-incubation with target cells, all secreted a certain level of IFN-γ cytokine, suggesting the effector function potential of the anti-BCMA bivalent CAR-T cells prepared in this application.

[0943] Example 22. Targeted proliferation ability of anti-BCMA bivalent CAR-T cells

[0944] In this embodiment, MOLP8 cells with low BCMA expression were used as the activation target cells to detect the targeted proliferation capacity of CAR-T cells. CAR-T cells activated by CD3 / CD28 DynaBeads and control Mock T cells were infected with lentivirus and their targeted proliferation capacity was detected approximately Day 9 of culture. As shown in Figures 46, 47, and 48, the sustained proliferation and killing capacity of bivalent BCMA CAR-T cells differed under multiple rounds of stimulation by either the BCMA-high expression target cell line MM.1S or the BCMA-low expression target cell line MOLP8. Overall, the bivalent BCMA CAR-T cells with the following combinations showed superior sustained proliferation and killing capacity: 3.2-(G4S)3-29, 29-(G4S)3-3.2, 11-(G4S)3-29, 29-(G4S)1-3.2, 29-(G4S)1-11, 1-(G4S)3-9.2, and 11-(G4S)3-3.2.

[0945] Example 23. In vivo efficacy experiment of anti-BCMA bivalent CAR-T cells in mice

[0946] To simulate the distribution of hematologic malignancies in the human body, and based on in vivo imaging technology, the tumor-reducing and tumor-suppressing effects of bivalent BCMA-specific CAR-T cells were verified in B-NSG mice. Specifically, the experiment included the following groups: a Mock T cell group and a bivalent BCMA-CAR-T cell group. B-NSG mice were injected with MOLP8-Luciferase target cells via the tail vein and randomly divided into groups of 5 mice each according to the bioluminescence intensity. A single dose of pre-frozen and thawed bivalent CAR-T or Mock T cells was injected via the tail vein. After CAR-T infusion, changes in bioluminescence in the mice were observed every Tuesday and Thursday using an animal in vivo imaging system, and the weight and health status of the mice were also observed.

[0947] As shown in Figures 49 and 50, compared with Mock T, bivalent BCMACAR-T cryopreserved cells showed significant anti-tumor activity, greatly reducing the tumor burden in mice, while the mice maintained normal weight and condition, indicating the safety of CAR-T cells.

[0948] Example 24. Design of Multispecific Chimeric Antigen Receptors

[0949] To obtain multispecific chimeric antigen receptor T-cell products with higher intercellular affinity and antitumor activity, we conducted in vitro and in vivo functional validation, selecting antibody sequences such as NanoBCMA1, NanoBCMA3.2, NanoBCMA9.2, NanoBCMA11, or NanoBCMA29 with high specificity, high target cell killing activity, high targeting proliferation capacity, and low tonic signaling levels. We constructed monovalent or bivalent BCMA-targeting CARs using (GGGGS)n (n = 1-5) linkers; and selected 3B5V... H H, 3E7V H H and 4B3V H Antibody sequences such as H are used to construct monovalent or bivalent binding domain GPRC5D-targeting CARs via (GGGGS)n (n = 1 to 5) linkers; the 383F12 antibody sequence is selected to construct a monovalent binding domain CD38-targeting CAR. This embodiment constructs a multispecific chimeric antigen receptor simultaneously targeting BCMA, GPRC5D, and CD38. The chimeric antigen receptor contains a CD8α signal peptide sequence (SP), a single-domain antibody against BCMA, a single-domain antibody against GPRC5D, a single-domain antibody against CD38, a (G4S)n linker (n = 1-5), a hinge region of CD8α or CD28, a transmembrane region sequence of CD8α or CD28 (TM), a 4-1BB or CD28 co-stimulatory domain sequence and a CD3ζ signal transduction domain sequence, a low-density lipoprotein receptor-associated protein or its fragment Ori. Schematic diagrams of the multispecific chimeric antigen receptor structure are shown in Figures 51-58, where the TanM series is consistent with the TriM series.

[0950] Example 25. Expression of GPRC5D, BCMA and CD38 antigens on the surface of target cells

[0951] This embodiment uses the DepMap Data Explorer database (https: / / depmap.org / portal / ) to predict the expression levels of BCMA, GPRC5D, and CD38 antigens on the surface of human myeloma cell lines NCIH929 and MOLP8. Flow cytometry was used to detect the actual expression levels of these cellular antigens. The positive rate of target cell antigen expression was detected by staining with PE-anti-human BCMA, APC-anti-human GPRC5D, and PE-anti-human CD38 antibodies and then by BD flow cytometry.

[0952] As shown in Figure 59, according to predictions from the DepMap Data Explorer database, the NCIH929 and MOLP8 cell lines specifically express GPRC5D, BCMA, and CD38, with differences in expression abundance. As shown in Figure 60, the negative control cells generally do not express BCMA, GPRC5D, and CD38. The human myeloma cell lines NCIH929 and MOLP8 specifically express BCMA and CD38, with differences in expression abundance; specifically, the BCMA antigen expression abundance on the surface of NCIH929 cells is higher than that of MOLP8. Furthermore, the GPRC5D antigen expression positivity rate of the NCIH929 cell line is low, while the expression level of MOLP8 is close to negative.

[0953] Example 26. Construction of a multispecific chimeric antigen receptor lentiviral expression vector

[0954] The construction of the multispecific chimeric antigen receptor lentiviral expression vector is briefly described as follows: The CAR lentiviral core empty vector plasmid (self-constructed by Yuanqi Biotechnology, hereinafter referred to as the CAR lentiviral empty vector) was double-digested with SphI (Cat.#R3182L, NEB) and NotI Cat.#R3189L, NEB to generate a linearized fragment; Nanjing Genscript Biotech Co., Ltd. synthesized a signal peptide sequence (SP) encoding CD8α from the N-terminus to the C-terminus; single-domain antibodies against BCMA NanoBCMA3.2 and / or NanoBCMA11 and / or NanoBCMA29; and single-domain antibodies against GPRC5D... The following nucleic acid sequences were cloned into a linearized lentiviral vector and transformed into DH5α competent cells: 3E7VHH and / or 4B3VHH and / or 3B5VHH; anti-CD38 single-domain antibody 383F12; the hinge region of CD8α or CD28, the transmembrane region (TM) sequence of CD8α or CD28, the co-stimulatory domain sequence of 4-1BB or CD28, and the CD3ζ signal transduction domain sequence; and the nucleic acid sequence of the CAR backbone polypeptide of low-density lipoprotein receptor-associated protein or its fragment Ori. These sequences were then cloned into a linearized lentiviral vector and transformed into DH5α competent cells. Single colonies were picked and sequenced for identification. The cloned multispecific chimeric antigen receptor lentiviral expression vector with correct sequencing results underwent plasmid extraction for subsequent lentiviral packaging.

[0955] Example 27. Packaging and titer determination of multispecific chimeric antigen receptor lentiviruses

[0956] The specific experimental methods for packaging and titer determination of the multispecific chimeric antigen receptor lentivirus are as described in Example 11. Viral titers were evaluated by flow cytometry, as shown in Figure 61. The exemplary multispecific chimeric antigen receptor lentivirus effectively infected 293T cells. The positive expression ratios of BCMA CAR and CD38 CAR were similar, indicating that the 2A self-cleaving peptide has high cleavage efficiency. The titer range of the multispecific CAR lentivirus prepared in this application is 2-10 × 10⁻⁶. 6 TU / mL can be used for CAR-T cell function verification.

[0957] Example 28. Lentiviral infection and preparation of multispecific CAR-T cells

[0958] As an example, the preparation method of CAR-T cells containing BCMA, GPRC5D and CD38 multispecific nanobody sequences and positive control sequences LCAR-B38M (patent CN109153731B), OriC321 (patent WO2023169555A9), and Ori017 (patent WO2023134718A1) is as follows:

[0959] In this embodiment, CD3 microbeads reagent kit (Miltenyi Biotec, Cat. #130-097-043) was used to sort CD3 in PBMCs. + T cells, in short, were revived via PBMC (Ficoll density gradient centrifugation), centrifuged at 500g for 5 minutes to remove cryopreservation medium; then 1 x 10 7 Resuspend the cells in 80 μL of pre-cooled sorting buffer; adjust the ratio to 1 x 10⁻⁶ cells / mL. 7 Add 20 μL of LCD3 MicroBeads to the total cell count, mix thoroughly, and incubate at 4°C for 15 min; after incubation, add 1 x 10⁻⁶ cells. 7 Add 1.5 mL of pre-cooled sorting buffer to the total cell count, centrifuge at 500 g for 5 min and discard the supernatant; then add 1 x 10⁻⁶ cells. 7 The total cell count was resuspended in 0.1 mL of room temperature separation buffer, mixed by pipetting, and the cell suspension was added to a rinsed LS separation column (placed in a magnetic field) to collect CD3+. + T cells were thoroughly mixed, centrifuged at 500g for 5 minutes to remove the supernatant, and the cell density was adjusted to 1.25 x 10⁶ cells / mL with X-VIVO complete medium. 6 T cells were activated by adding CD3 / CD28 DynaBeads at a cell / bead ratio of 1:3 and cultured at 37°C with 5% CO2. Flow cytometry analysis confirmed that PBMCs, after CD3-positive magnetic bead sorting, yielded CD3-positive DynaBeads with a purity >95%. + T cells.

[0960] T cells activated for 24 hours were collected in 50 mL centrifuge tubes, centrifuged at 500g for 5 min, the supernatant was removed, and the cells were resuspended in X-VIVO complete medium for counting. Cells were counted at a ratio of 7 x 10⁻⁶. 5 The total number of cells was determined, and the cells were seeded into 12-well plates with a multiplicity of infection (MOI) of 5. The required volume of lentivirus was added, and polybrene reagent was added to a final concentration of 10 μg / mL. The mixture was thoroughly mixed, centrifuged at 1200 rpm for 60 min, and cultured at 37°C with 5% CO2. After 24 h, the cells were collected, centrifuged at 500 g for 5 min to remove the supernatant, and resuspended in X-VIVO complete medium. The cell density was adjusted to 7 x 10⁶ cells / well. 5 The cells / mL were mixed and seeded into 12-well cell culture plates and cultured at 37°C with 5% CO2.

[0961] Example 29. Detection of CAR gene expression efficiency and in vitro amplification in multispecific CAR-T cells

[0962] The specific experimental methods for CAR gene expression efficiency and in vitro amplification detection are as described in Example 13. Results showed that when the lentiviral infection multiplicity MOI was 5, the CAR gene was stably expressed in multispecific CAR-T cells from different PBMC sources, and the CAR gene in T cells... + The transduction efficiency ranged from 20% to 85.19%, with an overall upward trend in the positive rate, which can be used for subsequent CAR-T cell function verification. As shown in Figure 62, the total expansion fold of multispecific CAR-T cells after activation and culture for 11 days was between 60 and 150 times; as shown in Figure 63, the viability of multispecific CAR-T cells after activation and culture for 12 days increased with the extension of culture time, and the overall cell viability remained above 80%.

[0963] Example 30. Evaluation of the in vitro targeted killing activity of multispecific CAR-T cells

[0964] CAR-T cells activated by CD3 / CD28 DynaBeads were cultured for 9 days, and their effector killing activity was assessed using the Luciferase Assay System kit (Cat.#E6120, Promega). In summary, the total number of target cells expressing the Luciferase gene was 3.3 x 10⁻⁶. 4Cells were seeded per well in a 96-well plate. The prepared CAR-T cells were pre-tested for positivity. Effector cells were mixed with target cells at ratios of 3:1, 1:1, and 1:3 (E:T = 3:1, 1:1, 1:3), with three replicates per group. The cells were co-cultured at 37°C in a 5% CO2 incubator for 24 hours. After co-culture, the cells were thoroughly mixed, and 100 μL of the cell suspension was transferred to an opaque 96-well plate. 100 μL of Luciferase Assay Substrate was added, mixed thoroughly, and incubated at room temperature in the dark for 5 minutes. The luciferase luminescence intensity in the remaining target cells was measured using a multi-functional microplate reader. The killing efficiency was quantitatively assessed using the luciferase in the remaining tumor cells after killing.

[0965] Formula for calculating cell kill ratio:

[0966] As shown in Figures 64-69, in T cells derived from different PBMCs, under conditions of added / unadded free sBCMA protein (20000 ng / mL), compared with blank effector T cells, the results showed that multispecific CAR-T cells could specifically kill target cells NCIH929-Luci-puro and MOLP8-Luci-puro. Furthermore, the exemplary multispecific CAR-T cells exhibited stronger effector activity and were less affected by free sBCMA compared to LCAR-B38M and OriC321 positive control CAR-T cells. Figure 70 shows the effect of exemplary multispecific CAR-T cells on the CD38 target antigen-positive target cell line NCIH929-BCMA. KO GPRC5D KO -CD38 Pos -Luci-puro does not exhibit non-specific killing. Overall, multispecific CAR-T cells showed relatively good killing effects, with a killing efficiency approaching 95% at an effector-to-target ratio of 1:1. The addition of free sBCMA protein to the co-incubation system may block the binding of anti-BCMACAR-T cells to the BCMA receptor on the surface of tumor cells and reduce CAR-T functional activity, while multispecific CAR-T cells still maintained high killing activity.

[0967] Example 31. Assessment of Multispecific CAR-T Cytokine Secretion Levels

[0968] This embodiment aims to detect the cytokine secretion levels of BCMA, GPRC5D, and / or CD38 multispecific CAR-T cells co-incubated with positive target cells in the presence and absence of free BCMA protein. Exemplary multispecific CAR-T cells were cultured in vitro for Day 9, and the levels of CAR-T cytokines IL-2 and IFN-γ were detected using an IL-2 and IFN-γ cytokine ELISA kit from R&D Corporation. The positivity rate and cell density of CAR-T cells were pre-tested. Lentivirally infected CAR-T cells were adjusted with blank T cells to ensure a consistent total cell count across groups. CAR-T cells and MOLP8 target cells were co-incubated at an effector-to-target ratio of 1:1, with the total number of CAR-positive cells set at 3.3 × 10⁻⁶. 4 The total number of MOLP8 cells was 3 × 10⁻⁶. 4 Each group was set up with 3 replicates; the CAR-T cell group containing free BCMA protein was incubated with an appropriate concentration of BCMA-his protein for 1-2 hours in advance, and the rest of the operation was the same. After incubation for 24 hours, the cells were centrifuged at 250g for 5 minutes, and the supernatant was collected. The cytokine secretion was detected by the corresponding IL-2 and IFN-γ ELISA kits.

[0969] As shown in Figures 71-73, in NCIH929, NCIH929-BCMA KO Under stimulation with MOLP8 target cells, the levels of IL-2 and IFN-γ cytokine secretion in multispecific CAR-T cells were higher than those in the Mock T cell group. IFN-γ secretion in all CAR-T groups was significantly higher than in the Mock T cell group. Furthermore, compared to the positive control LCAR-B38M and OriC321 CAR-T groups, multispecific CAR-T cells exhibited comparable or even higher levels of IL-2 and IFN-γ secretion, indicating a strong antigen-specific activation effect. In addition, in the presence of free sBCMA protein, multispecific CAR-T cells maintained a relatively high level of cytokine secretion compared to the positive control CAR-T cells, suggesting that multispecific CAR-T cells can alleviate the inhibitory effect of free sBCMA on CAR-T activity while maintaining antigen-specific activation and cytokine secretion.

[0970] Example 32. Assessment of Multispecific CAR-T Cell Targeted Proliferation Capacity

[0971] In this embodiment, untreated MOLP8-Luci cells were used as target cells for the detection of the targeted proliferation capacity of multispecific CAR-T cells. CD3 / CD28 DynaBeads-activated T cells were infected with lentivirus to prepare CAR-T cells and control Mock T cells. The targeted proliferation capacity of CAR-T cells was detected according to the culture time of Day 9. In short, the CAR positivity rate of each group was detected in advance, and blank T cells were used to adjust the CAR positivity rate of each group to be consistent (the culture medium was X-VIVO without any additives). 15 (Blank culture medium). CAR-T cells and MOLP8-Luci cells were subjected to the first round of targeted stimulation at an effector-to-target ratio of 1:1, with each round lasting 24 hours, for a total of 6 rounds. In rounds 2-6, the same number of target cells were added each round. After each round, the cells in the culture system were thoroughly mixed, and cell counts were performed using AO / PI reagents. Cell density and cell diameter were recorded. Half of the cell volume was then co-incubated with fresh target cells for a new round of targeted stimulation. After each round, the cytotoxic effector activity of multispecific CAR-T cells, the proportion of residual target cells, the CAR-T cell positivity rate, and the abundance of CAR molecule expression were measured.

[0972] Figure 74 shows that the exemplary multispecific CAR-T cells exhibited superior sustained killing ability during co-incubation with different target cells, and were no weaker than LCAR-B38M positive control CAR-T cells. Figure 75 shows that during repeated stimulation by target cells, the CAR gene expression level of the exemplary multispecific CAR-T cells remained relatively stable or showed an increasing trend. Figure 76 shows that during repeated stimulation by target cells, the CAR molecule expression abundance of the exemplary multispecific CAR-T cells was upregulated under initial target cell stimulation and decreased with multiple rounds of target cell stimulation, which is beneficial for maintaining CAR functional stability. Figure 77 shows that during repeated stimulation by target cells, the exemplary multispecific CAR-T cells exhibited varying degrees of targeted proliferation ability, with a proliferation fold of 50-100 times after 6 rounds of repeated stimulation, compared with the control Mock. The significant differences compared to T cells suggest that multispecific CAR-T cells can effectively and rapidly expand in vivo against tumors with low to medium levels of target antigen expression, indicating their potential for sensitivity, effectiveness, and rapid expansion against specific tumor antigens in vivo. As shown in Figures 78-79, during repeated stimulation of target cells, exemplary multispecific CAR-T cells exhibited different levels of IFN-γ and IL-2 cytokine release, revealing the functional activity of CAR-T cells under target cell stimulation.

[0973] Example 33. IL-6 secretion level in a multispecific CAR-T cell co-incubation system with monocytes and target cells.

[0974] CRS (Cytokine Release Syndrome) is caused by the secretion of large amounts of pro-inflammatory cytokines by macrophages or monocytes in response to IFN-γ and possibly related cytokines secreted by CAR-T cells. Studies have found that monocytes are the primary cells mediating CRS by releasing IL-6; therefore, this experiment evaluated the release of IL-6 from CAR-T cells co-cultured with target cells. In short, CD14+ monocytes (M) were isolated from PBMCs using the CD14 MicroBeads kit (Cat.#130-050-201, Miltenyi Biotech), and CD3+ T cells were sorted from the same healthy donor PBMCs for CAR-T production using the CD3 MicroBeads kit (Cat.#130-097-043, Miltenyi Biotech). CAR-T cells cultured alone, target cells, or monocytes were used as negative controls. Multispecific CAR-T cells were co-cultured with target cells and monocytes at an E:T:M ratio of 3:1:1, or with target cells at an E:T ratio of 3:1, or with monocytes at an E:M ratio of 3:1 for 24 hours. The culture medium was then harvested for cytokine quantification and analysis using a human IL-6 kit (Cat.#62HIL06PEG, Cisbio).

[0975] As shown in Figure 80, in the co-culture system of multispecific CAR-T cells, target cells, and monocytes, both multispecific CAR-T cells and the positive control LCAR B38M CAR-T cells induced monocytes to release low levels of IL-6, indicating a low potential risk of CRS. Furthermore, IL-6 release was undetectable in the co-culture of multispecific CAR-T cells and monocytes, suggesting that without specific antigen stimulation, multispecific CAR-T cells do not induce monocytes to release IL-6, further indicating a low potential risk of CRS.

[0976] Example 34. Study on multispecific CAR-T cell subtypes and phenotypes

[0977] This embodiment uses flow cytometry (FACS) to detect multispecific CAR-T cell phenotypes, providing important immune monitoring indicators for CAR-T cell therapy. In short, CD3+ activation after lentiviral infection... + CAR-T cells were prepared from T cells. After obtaining positive CAR expression detection, the memory phenotype of the cells was detected on Day 9 of culture: effector CAR-T cells corresponding to the corresponding culture days were collected, and an appropriate number of cells were used for flow cytometry to detect CD3 in each group. + T cell subtype markers: including CD3-PB, CD4-APC, CD8-PerCP-Cy5.5; detection of CD3 in each group+ Memory markers in T cells: including the proportion of CD4-APC, CD8-PerCP-Cy5.5, CD45RA-BV510, CCR7-PE, and CD95-PECY7 cells, for activated T cells (CD3+). + CD95 + CD45RA and CCR7 can be used as the minimum marker combination to distinguish memory T cells at different stages, specifically classifying them into T cells. SCM (CD45RA + CCR7 + ), T CM (CD45RA - CCR7 + ), T EM (CD45RA - CCR7 - ), T EFF (CD45RO + CCR7 - ); Detect CD3 in each group + Activating markers in T cells: including the proportion of CD3-BV510, CD137-PE, CD25-PE-CY7, and CD69-APC cells; detection of CD3 in each group. + Exhaustion markers in T cells: including the proportion of CD3-PerCP-Cy5.5, PD-1-BV421, LAG-3-AF647, and TIM-3-BV510 cells.

[0978] As shown in Figure 81, on day 12 of CAR-T cell culture, pcM2-Ori2 CAR-T cells exhibited a higher stem cell memory (TSCM) phenotype compared to other structures, suggesting that the pcM2-Ori2 CAR structure helps maintain a lower differentiation state of T cells and retain more early memory features, which may endow CAR-T cells with stronger proliferative potential and durable anti-tumor function in vivo.

[0979] Example 35. Construction of a bispecific chimeric antigen receptor lentiviral expression vector

[0980] The construction of the bispecific chimeric antigen receptor and control BMS Dual G5xBCMA (BMS-986453) lentiviral expression vector is briefly described as follows: The CAR lentiviral core empty vector plasmid was double-digested with SphI and NotI restriction endonucleases to generate a linearized fragment; Nanjing Genscript Biotech Co., Ltd. synthesized the signal peptide sequence (SP) encoding CD8α from the N-terminus to the C-terminus; the single-domain antibody 3B5VHH against GPRC5D; the single-domain antibody NanoBCMA3.2 and / or NanoBCMA11 and / or NanoBCMA29 against BCMA; the hinge region of CD8α or CD28, the transmembrane region sequence (TM) of CD8α or CD28, the co-stimulatory domain sequence of 4-1BB or CD28 and the CD3ζ signal transduction domain sequence, and the nucleic acid sequence of the CAR backbone polypeptide of low-density lipoprotein receptor-associated protein or its fragment Ori. The nucleic acid sequence was cloned into the linearized lentiviral vector and transformed into DH5α competent cells. Single colonies were picked and sequenced for identification. The correctly sequenced bispecific chimeric antigen receptor lentiviral expression vector clones were subjected to plasmid extraction for subsequent lentiviral packaging, as shown in Figure 82. The structure contains tandem and parallel chimeric co-stimulatory receptor (CCR) forms.

[0981] Example 36. Packaging and titer determination of bispecific chimeric antigen receptor lentivirus

[0982] The specific experimental methods for packaging and titer determination of the bispecific chimeric antigen receptor lentivirus are as described in Example 11. Viral titers were evaluated by flow cytometry, and the results showed that the titer range of the bispecific CAR lentivirus prepared in this application was 2-10 × 10⁻⁶. 6 TU / mL can be used for CAR-T cell function verification.

[0983] Example 37. Lentiviral infection and preparation of bispecific CAR-T cells

[0984] As an example, the preparation method of CAR-T cells containing GPRC5D, BCMA bispecific nanobody sequences is as follows: CD3 in PBMCs is sorted using a CD3 MicroBeads kit. + T cells, in short, were revived via PBMC (Ficoll density gradient centrifugation), centrifuged at 500g for 5 minutes to remove cryopreservation medium; then 1 x 10 7 Resuspend the cells in 80 μL of pre-cooled sorting buffer; adjust the ratio to 1 x 10⁻⁶ cells / mL. 7 Add 20 μL of CD3 MicroBeads to the total cell count, mix thoroughly, and incubate at 4°C for 15 min; after incubation, add 1×10⁻⁶ cells. 7 Add 1.5 mL of pre-cooled sorting buffer to the total cell count, centrifuge at 500 g for 5 min and discard the supernatant; then add 1 × 10⁻⁶ cells. 7The total cell count was resuspended in 0.1 mL of room temperature separation buffer, mixed by pipetting, and the cell suspension was added to a rinsed LS separation column (placed in a magnetic field) to collect CD3+. + T cells were thoroughly mixed, centrifuged at 500g for 5 minutes to remove the supernatant, and the cell density was adjusted to 1.25 × 10⁶ cells / mL using X-VIVO complete medium. 6 T cells were activated by adding CD3 / CD28 DynaBeads at a cell / bead ratio of 1:3 and cultured at 37°C with 5% CO2. Flow cytometry analysis confirmed that PBMCs, after CD3-positive magnetic bead sorting, yielded CD3-positive DynaBeads with a purity >95%. + T cells.

[0985] T cells activated for 24 hours were collected in 50 mL centrifuge tubes, centrifuged at 500g for 5 min, the supernatant was removed, and the cells were resuspended in X-VIVO complete medium for counting. Cells were counted at a rate of 7 × 10⁻⁶. 5 The total number of cells was determined, and the cells were seeded into 12-well plates with a multiplicity of infection (MOI) of 5. The required volume of lentivirus was added, and polybrene reagent was added to a final concentration of 10 μg / mL. The mixture was thoroughly mixed, centrifuged at 1200 rpm for 60 min, and cultured at 37°C with 5% CO2. After 24 h, the cells were collected, centrifuged at 500 g for 5 min to remove the supernatant, and resuspended in X-VIVO complete medium to adjust the cell density to 7 × 10⁶ cells / well. 5 The cells / mL were mixed and seeded into 12-well cell culture plates and cultured at 37°C with 5% CO2.

[0986] Example 38. Detection of CAR gene expression efficiency and in vitro amplification in bispecific CAR-T cells.

[0987] The specific experimental methods for CAR gene expression efficiency and in vitro amplification detection are as described in Example 13. As shown in Figure 83, the results indicate that when the lentiviral infection multiplicity MOI = 5, the CAR gene is stably expressed in bispecific CAR-T cells from T cells derived from different PBMCs, and the CAR gene in T cells... + With a transduction efficiency of over 65%, it can be used for subsequent CAR-T cell function validation.

[0988] Example 39. Evaluation of the in vitro targeted killing activity of bispecific CAR-T cells

[0989] An in vitro model of multiple myeloma (MM) cells was used to detect CAR-T cell killing of MM cells resembling those from early, intermediate, and advanced stages of the disease. Specifically, different MM cell lines were used, with high / medium / low expression levels of the dual or single antigens GPRC5D or BCMA on their surface, to reflect the antigen abundance characteristics of different MM subsets and different disease stages or previously treated myeloma cells. To screen for CAR binders resistant to soluble BCMA inhibition, free BCMA was added to the CAR-T cell-tumor cell co-incubation system in vitro. In summary, the total number of target cells expressing the Luciferase gene was 3.3 × 10⁻⁶. 4 Cells were seeded per well in a 96-well plate. The prepared CAR-T cells were pre-tested for positivity. Effector cells were mixed with target cells at ratios of 3:1, 1:1, and 1:3 (E:T = 3:1, 1:1, 1:3), with three replicates per group. The cells were co-cultured at 37°C in a 5% CO2 incubator for 24 hours. After co-culture, the cells were thoroughly mixed, and 100 μL of the cell suspension was transferred to an opaque 96-well plate. 100 μL of Luciferase Assay Substrate was added, mixed thoroughly, and incubated at room temperature in the dark for 5 minutes. The luciferase luminescence intensity in the remaining target cells was measured using a multi-functional microplate reader. The killing efficiency was quantitatively assessed using the luciferase in the remaining tumor cells after killing.

[0990] Formula for calculating cell kill ratio:

[0991] As shown in Figures 84-86, the results indicate that, regardless of the presence or absence of high concentrations of sBCMA, the bispecific CAR-T cells exhibited superior killing activity against multiple myeloma cells (H929 cell line: BCMA high, G5 med; MM.1S cell line: BCMA med, G5 low; MOLP.8 cell line: BCMA very low, G5 negative) compared to the competing dual-target BMS G5×BCMA scFv CAR-T (derived from WO2024031091A2, with a tandem loop structure, also known as "BMSDual G5×BCMA CAR-T"), demonstrating a better targeted killing effect. Among them, CAR-T cells containing the ori element showed the best killing activity. As shown in Figure 87, the results indicate that the bispecific CAR-T constructed in this application showed higher sensitivity to changes in BCMA antigen abundance. In the low BCMA abundance cell model (MOLP-8), the killing activity of both tandem and parallel CAR-T structures was significantly better than that of the control competing CAR-T.

[0992] Example 40. Assessment of Bispecific CAR-T Cytokine Secretion Levels

[0993] This embodiment aims to detect the cytokine secretion levels of BCMA, GPRC5D bispecific CAR-T cells co-incubated with positive target cells in the presence and absence of free BCMA protein. Exemplary bispecific CAR-T cells were cultured in vitro for Day 9, and the levels of CAR-T cytokines IL-2 and IFN-γ were detected using an IL-2 and IFN-γ cytokine ELISA kit from R&D Corporation. The positivity rate and cell density of CAR-T cells were pre-tested. Lentivirally infected CAR-T cells were adjusted with blank T cells to ensure a consistent total cell count across groups. CAR-T cells and MOLP8 target cells were co-incubated at an effector-to-target ratio of 1:1, with the total number of CAR-positive cells set at 3.3 × 10⁻⁶. 4 The total number of MOLP8 cells was 3 × 10⁻⁶. 4 Each group was set up with 3 replicates; the CAR-T cell group containing free BCMA protein was incubated with an appropriate concentration of BCMA-his protein for 1-2 hours in advance, and the rest of the operation was the same. After incubation for 24 hours, the cells were centrifuged at 250g for 5 minutes, and the supernatant was collected. The cytokine secretion was detected by the corresponding IL-2 and IFN-γ ELISA kits.

[0994] As shown in Figures 88 and 89, under stimulation by NCIH929, MM1S, and MOLP8 target cells, the IL-2 and IFN-γ cytokine secretion levels of bispecific CAR-T cells were higher than those of the Mock T cell group. The IFN-γ secretion in each CAR-T group was significantly higher than that in the Mock T cell group. Furthermore, compared to the positive control competitor's dual-target G5×BCMA scFv CAR-T group, bispecific CAR-T cells exhibited comparable or even higher IL-2 and IFN-γ secretion levels, indicating a strong antigen-specific activation effect. In addition, in the presence of free sBCMA protein, bispecific CAR-T cells maintained a relatively high cytokine secretion level compared to the positive control CAR-T cells, suggesting that bispecific CAR-T cells can alleviate the inhibitory effect of free sBCMA on CAR-T activity while maintaining antigen-specific activation and cytokine secretion.

[0995] Example 41. Assessment of the bispecific CAR-T cell targeting and proliferation capacity

[0996] In this embodiment, the targeting and proliferation capacity of bispecific CAR-T cells was detected using untreated MM.1S, NCIH929, and NCIH929-BCMA. KO NCIH929-GPRC5D KOCells were used as target cells. CD3 / CD28 DynaBeads-activated T cells were infected with lentivirus to prepare CAR-T cells and control Mock T cells. The CAR-T cell targeting and proliferation capacity was assessed according to the culture time (Day 9). In short, the CAR positivity rate of each group was pre-tested, and blank T cells were used to adjust the CAR positivity rate of each group to be consistent (the culture medium was X-VIVO without any additives). 15 (Blank culture medium). CAR-T cells and target cells were subjected to the first round of targeted stimulation at an effector-to-target ratio of 1:1, with each round lasting 24 hours, for a total of 6 rounds. In rounds 2-6, the same number of different target cells were added each round. After each round, the cells in the culture system were thoroughly mixed, and cell counts were performed using AO / PI reagents. Cell density and cell diameter were recorded. Half of the cell volume was then co-incubated with fresh target cells for a new round of targeted stimulation. After each round, the cytotoxic effect activity of multispecific CAR-T cells, the proportion of residual target cells, the CAR-T cell positivity rate, and the abundance of CAR molecule expression were measured.

[0997] As shown in Figure 90, the bispecific CAR-T cells prepared in this application exhibited superior sustained killing ability during co-incubation with different target cells, and were no weaker than the B38MBBZ positive control CAR-T cells. The CAR gene expression level of the bispecific CAR-T cells was relatively stable. In addition, the CAR-T cells had low levels of autocrine cytokines when not co-incubated with target cells, indicating that the spontaneous activation (tonic signaling) of the bispecific CAR-T cells was at baseline.

[0998] Example 42. Verification of Bispecific CAR-T under Reduction Conditions and Low pH Interference

[0999] This study aimed to verify the stability...

Claims

1. An isolated antigen-binding protein capable of binding B cell maturation antigen (BCMA), said isolated antigen-binding protein comprising at least one CDR in the variable region VH of the antibody heavy chain, said VH comprising the amino acid sequence shown in any one of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:

72.

2. The antigen-binding protein according to claim 1, comprising an antibody or an antigen-binding fragment thereof.

3. The antigen-binding protein according to any one of claims 1-2, wherein the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

4. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding fragment is VHH.

5. The antigen-binding protein according to any one of claims 1-4, wherein the antibody comprises a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or a fully human antibody.

6. The antigen-binding protein according to any one of claims 1-5, comprising HCDR3, wherein the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:

15.

7. The antigen-binding protein according to any one of claims 1-6, comprising HCDR3, wherein said HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:

15.

8. The antigen-binding protein according to any one of claims 1-7, comprising HCDR2, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:20 or SEQ ID NO:

23.

9. The antigen-binding protein according to any one of claims 1-8, comprising HCDR2, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:

23.

10. The antigen-binding protein according to any one of claims 1-9, comprising HCDR1, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:31, SEQ ID NO:28, SEQ ID NO:27 or SEQ ID NO:

32.

11. The antigen-binding protein according to any one of claims 1-10, comprising HCDR1, wherein the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:

30.

12. The antigen-binding protein according to any one of claims 1-11, comprising HCDR1, HCDR2, and HCDR3, and said antigen-binding protein comprising an amino acid sequence selected from any one of the following groups: (1) HCDR1: SEQ ID NO: 31, HCDR2: SEQ ID NO: 24, and HCDR3: SEQ ID NO: 16; (2) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 25, and HCDR3: SEQ ID NO: 17; (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:

15.

13. The antigen-binding protein according to any one of claims 1-12, comprising HCDR1, HCDR2, and HCDR3, and said antigen-binding protein comprising an amino acid sequence selected from any one of the following groups: (1) HCDR1: SEQ ID NO: 26, HCDR2: SEQ ID NO: 19, and HCDR3: SEQ ID NO: 1; (2) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 2; (3) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 2; (4) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 3; (5) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 21, and HCDR3: SEQ ID NO: 4; (6) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 5; (7) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 6 (8) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 7; (9) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 8; (10) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 9; (11) HCDR1: SEQ ID NO: 28, HCDR2: SEQ ID NO: 22, and HCDR3: SEQ ID NO: 10; (12) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 11; (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 12; (14) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 13; (15) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 14; (16) HCDR1: SEQ ID NO:29, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:15; and (17) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:23, and HCDR3: SEQ ID NO:

15.

14. The antigen-binding protein according to any one of claims 1-13, wherein the variable region of the antibody heavy chain comprises the amino acid sequence shown in any one of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:

72.

15. The antigen-binding protein according to claims 1-14, wherein the antibody heavy chain variable region is VHH, and the VHH comprises the amino acid sequence shown in any one of SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, and SEQ ID NO:

72.

16. The antigen-binding protein according to any one of claims 1-15, comprising an antibody heavy chain constant region derived from IgG.

17. The antigen-binding protein according to any one of claims 1-16, comprising an antibody heavy chain constant region derived from human IgG.

18. The antigen-binding protein according to any one of claims 1-17, comprising an antibody heavy chain constant region derived from human IgG1.

19. A chimeric antigen receptor comprising a targeting portion, said targeting portion comprising at least one antigen-binding domain targeting BCMA, and said antigen-binding domain comprising the antigen-binding protein of any one of claims 1-18.

20. The chimeric antigen receptor of claim 19, comprising two BCMA-targeting antigen-binding domains, a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain are each independently selected from any one of the antigen-binding proteins of claims 1-18.

21. The chimeric antigen receptor of claim 20, wherein the first antigen-binding domain and the second antigen-binding domain target the same epitope of BCMA.

22. The chimeric antigen receptor of claim 20, wherein the first antigen-binding domain and the second antigen-binding domain target different epitopes of BCMA.

23. The chimeric antigen receptor according to claims 20-22, wherein the first antigen-binding domain and the second antigen-binding domain comprise the same or different amino acid sequences.

24. The chimeric antigen receptor according to claims 20-23, wherein the first antigen-binding domain and the second antigen-binding domain are directly or indirectly connected.

25. The chimeric antigen receptor according to claims 20-24, wherein the first antigen-binding domain and the second antigen-binding domain are connected by a linker.

26. The chimeric antigen receptor according to claim 25, wherein the linker is a linker peptide.

27. The chimeric antigen receptor of claim 26, wherein the linker peptide comprises the amino acid sequence (GGGGS)n, wherein n is any positive integer from 1 to 10.

28. The chimeric antigen receptor of claim 26, wherein the linker peptide comprises an amino acid sequence of (EAAAK)n, wherein n is any positive integer from 1 to 10.

29. The chimeric antigen receptor according to any one of claims 20-28, wherein the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain comprise amino acid sequences selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (3) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

18. (4) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:17, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (6) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17; (7) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:27, HCDR2 is SEQ ID NO:20, and HCDR3 is SEQ ID NO:18; (8) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:32, HCDR2 is SEQ ID NO:23, and HCDR3 is SEQ ID NO:15; (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18; (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (13) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (14) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (15) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18; and (16) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:

15.

30. The chimeric antigen receptor according to any one of claims 20-29, wherein the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain comprise amino acid sequences selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (3) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (4) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (6) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (7) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (8) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (13) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (14) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (15) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (16) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (17) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (18) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (19) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (20) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

2. (21) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:

2. (22) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (23) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (24) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (25) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (26) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (27) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (28) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (29) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

13. (30) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (31) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (32) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (33) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:

2. (34) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (35) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; and (36) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:

15.

31. The chimeric antigen receptor according to any one of claims 19-30, comprising a co-stimulatory signaling region, wherein the co-stimulatory signaling region comprises an intracellular co-stimulatory signaling region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

32. The chimeric antigen receptor of claim 31, wherein the co-stimulatory signal region is an intracellular co-stimulatory signal region derived from 4-1BB.

33. The chimeric antigen receptor according to any one of claims 19-32, comprising an intracellular signaling region comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

34. The chimeric antigen receptor of claim 33, wherein the intracellular signaling region is a signal transduction domain derived from CD3ζ.

35. The chimeric antigen receptor according to any one of claims 19-34, comprising a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

36. The chimeric antigen receptor of claim 35, wherein the transmembrane region is a transmembrane region derived from CD8.

37. The chimeric antigen receptor according to any one of claims 19-36, comprising a hinge region between the targeting portion and the transmembrane region, said hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

38. The chimeric antigen receptor of claim 37, wherein the hinge region is a hinge region derived from CD8.

39. The chimeric antigen receptor according to any one of claims 19-38, further comprising low-density lipoprotein receptor-associated protein or a fragment thereof, said low-density lipoprotein receptor-associated protein or a fragment thereof being located at the C-terminus of said intracellular signaling region.

40. The chimeric antigen receptor of claim 39, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments.

41. The chimeric antigen receptor according to any one of claims 40, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

42. The chimeric antigen receptor according to any one of claims 19-41, further comprising a signal peptide.

43. The chimeric antigen receptor according to claims 19-42, wherein the signal peptide is derived from the signal peptide of the CD8 protein.

44. A chimeric antigen receptor comprising a targeting portion, said targeting portion comprising at least a first targeting portion and a second targeting portion, wherein the first targeting portion targets BCMA and the second targeting portion targets GPRC5D.

45. The chimeric antigen receptor of claim 44, wherein the first targeting portion targeting BCMA comprises at least one antigen-binding domain targeting BCMA, and the antigen-binding domain comprises an antigen-binding protein of any one of claims 1-18, and / or a chimeric antigen receptor of any one of claims 19-43.

46. ​​The chimeric antigen receptor according to any one of claims 44-45, wherein the first targeting portion targeting BCMA comprises a first antigen-binding domain targeting BCMA and a second antigen-binding domain targeting BCMA.

47. The chimeric antigen receptor of claim 46, wherein the first antigen-binding domain targeting BCMA and the second antigen-binding domain targeting BCMA are each independently selected from any one of claims 1-18 of the antigen-binding protein.

48. The chimeric antigen receptor according to any one of claims 44-47, wherein the second targeting portion targets plasma cell malignant tumor antigens.

49. The chimeric antigen receptor of claim 48, wherein the second targeting portion targets the G protein-coupled receptor family.

50. The chimeric antigen receptor of claim 49, wherein the second targeting portion targets GPRC5D.

51. The chimeric antigen receptor of claim 50, wherein the second targeting portion targeting GPRC5D comprises at least one CDR in the variable region VH of the antibody heavy chain, the VH comprising SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:

87.

52. The chimeric antigen receptor of claim 51, wherein the second targeting portion targeting GPRC5D comprises HCDR3, and the HCDR3 comprises the amino acid sequences shown in SEQ ID NO:75, SEQ ID NO:78, and / or SEQ ID NO:

81.

53. The chimeric antigen receptor according to any one of claims 50-52, wherein the second targeting portion targeting GPRC5D comprises HCDR2, and the HCDR2 comprises the amino acid sequences shown in SEQ ID NO:74, SEQ ID NO:77, and / or SEQ ID NO:

80.

54. The chimeric antigen receptor according to any one of claims 50-53, wherein the second targeting portion targeting GPRC5D comprises HCDR1, and the HCDR1 comprises the amino acid sequences shown in SEQ ID NO:73, SEQ ID NO:76, and / or SEQ ID NO:

79.

55. The chimeric antigen receptor according to any one of claims 50-54, wherein the second targeting portion targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75; (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:

81.

56. The chimeric antigen receptor according to any one of claims 50-55, wherein the second targeting portion targeting GPRC5D comprises at least one antigen-binding domain targeting GPRC5D.

57. The chimeric antigen receptor of claim 56, wherein the second targeting portion targeting GPRC5D comprises an antigen-binding domain targeting GPRC5D.

58. The chimeric antigen receptor of claim 56, wherein the second targeting portion targeting GPRC5D comprises two antigen-binding domains targeting GPRC5D, a first antigen-binding domain targeting GPRC5D and a second antigen-binding domain targeting GPRC5D.

59. The chimeric antigen receptor of claim 58, wherein the first antigen-binding domain and the second antigen-binding domain target the same epitope of GPRC5D.

60. The chimeric antigen receptor of claim 58, wherein the first antigen-binding domain and the second antigen-binding domain target different epitopes of GPRC5D.

61. The chimeric antigen receptor according to any one of claims 58-60, wherein the first antigen-binding domain targeting GPRC5D and the second antigen-binding domain targeting GPRC5D comprise the same or different amino acid sequences.

62. The chimeric antigen receptor according to any one of claims 58-61, wherein the first antigen-binding domain targeting GPRC5D and the second antigen-binding domain targeting GPRC5D are directly or indirectly connected.

63. The chimeric antigen receptor according to any one of claims 58-62, wherein the first antigen-binding domain targeting GPRC5D and the second antigen-binding domain targeting GPRC5D are linked by a linker.

64. The chimeric antigen receptor according to claim 63, wherein the linker is a linker peptide.

65. The chimeric antigen receptor according to any one of claims 64, wherein the linker peptide comprises an amino acid sequence of (GGGGS)n, wherein n is any positive integer from 1 to 10.

66. The chimeric antigen receptor according to any one of claims 64, wherein the linker peptide comprises an amino acid sequence of (EAAAK)n, wherein n is any positive integer from 1 to 10.

67. The chimeric antigen receptor according to any one of claims 58-66, wherein the first antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D and HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D comprise amino acid sequences selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:

78. (3) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; (4) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:

78. (6) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; (7) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (8) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81, and the HCDR1 of the second antigen-binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; and (9) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:

81.

68. The chimeric antigen receptor according to any one of claims 50-56, wherein the second targeting portion targeting GPRC5D comprises three antigen-binding domains targeting GPRC5D.

69. The chimeric antigen receptor according to any one of claims 44-68, wherein the first targeting portion targeting BCMA is directly or indirectly connected to the second targeting portion targeting GPRC5D.

70. The chimeric antigen receptor according to any one of claims 44-69, wherein the first targeting portion targeting BCMA and the second targeting portion targeting GPRC5D are connected by a linker.

71. The chimeric antigen receptor of claim 70, wherein the linker comprises a linker peptide.

72. The chimeric antigen receptor of claim 71, wherein the linker peptide comprises an amino acid sequence of (GGGGS)n, wherein n is any positive integer from 1 to 10.

73. The chimeric antigen receptor of claim 71, wherein the linker peptide comprises an amino acid sequence of (EAAAK)n, wherein n is any positive integer from 1 to 10.

74. The chimeric antigen receptor according to any one of claims 44-73, wherein the first targeting portion targeting BCMA is connected to the second targeting portion targeting GPRC5D.

75. The chimeric antigen receptor according to any one of claims 44-74, wherein the first antigen-binding domain of the first targeting portion of BCMA is connected to the second targeting portion of GPRC5D, and the second targeting portion of GPRC5D is connected to the second antigen-binding domain of the first targeting portion of BCMA.

76. The chimeric antigen receptor according to any one of claims 44-75, wherein the first antigen-binding domain of the first targeting portion of BCMA is connected to the second antigen-binding domain of the first targeting portion of BCMA, and the second antigen-binding domain of the first targeting portion of BCMA is connected to the second targeting portion of GPRC5D.

77. The chimeric antigen receptor according to claims 44-76, further comprising a third targeting portion, said third targeting portion targeting plasma cell malignant tumor antigens.

78. The chimeric antigen receptor of claim 77, wherein the third targeting portion targets CD38.

79. The chimeric antigen receptor according to any one of claims 77-78, wherein the third targeting portion comprises at least one CDR in the variable region VH of the antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112 or SEQ ID NO:

110.

80. The chimeric antigen receptor according to any one of claims 77-78, wherein the third targeting portion targeting CD38 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

102.

81. The chimeric antigen receptor according to any one of claims 77-79, wherein the third targeting portion targeting CD38 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:90 or SEQ ID NO:

93.

82. The chimeric antigen receptor according to any one of claims 77-80, wherein the third targeting portion comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:96 or SEQ ID NO:

100.

83. The chimeric antigen receptor according to any one of claims 77-82, wherein the third targeting portion comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99 or SEQ ID NO:

97.

84. The chimeric antigen receptor according to any one of claims 77-83, wherein the third targeting portion targeting CD38 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:101 or SEQ ID NO:

88.

85. The chimeric antigen receptor according to any one of claims 77-84, wherein the third targeting portion targeting CD38 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:98 or SEQ ID NO:

94.

86. The chimeric antigen receptor according to any one of claims 77-85, wherein the third targeting portion targeting CD38 comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90; (2) HCDR1: SEQ ID NO:101, HCDR2: SEQ ID NO:100, and HCDR3: SEQ ID NO:102; and (3) HCDR1: SEQ ID NO: 101, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO:

102.

87. The chimeric antigen receptor according to any one of claims 77-86, wherein the third targeting portion targeting CD38 comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90; (2) HCDR1: SEQ ID NO:91, HCDR2: SEQ ID NO:92, and HCDR3: SEQ ID NO:93; (3) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:95, and HCDR3: SEQ ID NO:93; (4) HCDR1: SEQ ID NO:98, HCDR2: SEQ ID NO:99, and HCDR3: SEQ ID NO:93; and (5) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:97, and HCDR3: SEQ ID NO:

93.

88. The chimeric antigen receptor according to any one of claims 77-87, wherein the first targeting portion targeting BCMA, the second targeting portion targeting GPRC5D, and / or the third targeting portion targeting CD38 are directly or indirectly connected.

89. The chimeric antigen receptor according to any one of claims 77-88, wherein the first targeting portion targeting BCMA, the second targeting portion targeting GPRC5D, and / or the third targeting portion targeting CD38 are connected by a linker.

90. The chimeric antigen receptor of claim 89, wherein the linker comprises a linker peptide.

91. The chimeric antigen receptor of claim 90, wherein the linker peptide comprises the amino acid sequence (GGGGS)n, wherein n is any positive integer from 1 to 10.

92. The chimeric antigen receptor of claim 90, wherein the linker peptide comprises an amino acid sequence of (EAAAK)n, wherein n is any positive integer from 1 to 10.

93. The chimeric antigen receptor according to any one of claims 77-92, wherein the first targeting portion targeting BCMA, the second targeting portion targeting GPRC5D, and / or the third targeting portion targeting CD38 comprises an antibody or an antigen-binding fragment thereof.

94. The chimeric antigen receptor according to claim 93, wherein the antigen-binding fragment comprises Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH and / or dAb.

95. The chimeric antigen receptor of claim 94, wherein the antibody is selected from the group consisting of monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

96. The chimeric antigen receptor according to any one of claims 44-95, wherein the chimeric antigen receptor comprises a co-stimulatory signaling region, wherein the intracellular co-stimulatory signaling region comprises an intracellular co-stimulatory signaling region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

97. The chimeric antigen receptor according to any one of claims 44-96, wherein the chimeric antigen receptor comprises an intracellular signaling region comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

98. The chimeric antigen receptor according to any one of claims 44-97, wherein the chimeric antigen receptor comprises a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

99. The chimeric antigen receptor according to any one of claims 44-98, wherein the chimeric antigen receptor includes a hinge region between the targeting portion and the transmembrane region, the hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

100. A fusion protein comprising an extracellular region and a transmembrane region, wherein the extracellular region comprises at least one antigen-binding domain, the antigen-binding domain comprising the antigen-binding protein of any one of claims 1-18.

101. The fusion protein of claim 100, wherein the extracellular region comprises two BCMA-targeting antigen-binding domains, a first antigen-binding domain and a second antigen-binding domain, and the first antigen-binding domain and the second antigen-binding domain are each independently selected from the antigen-binding protein of any one of claims 1-18.

102. The fusion protein of claim 101, wherein the first antigen-binding domain and the second antigen-binding domain target the same epitope of BCMA.

103. The fusion protein of claim 101, wherein the first antigen-binding domain and the second antigen-binding domain target different epitopes of BCMA.

104. The fusion protein according to any one of claims 101-103, wherein the first antigen-binding domain and the second antigen-binding domain comprise the same or different amino acid sequences.

105. The fusion protein according to any one of claims 101-104, wherein the first antigen-binding domain and the second antigen-binding domain are directly or indirectly linked.

106. The fusion protein according to any one of claims 101-105, wherein the first antigen-binding domain and the second antigen-binding domain are linked by a linker.

107. The fusion protein according to claim 106, wherein the linker is a linking peptide.

108. The fusion protein of claim 107, wherein the linker peptide comprises the amino acid sequence (GGGGS)n, wherein n is any positive integer from 1 to 10.

109. The fusion protein of claim 107, wherein the linker peptide comprises the amino acid sequence (EAAAK)n, wherein n is any positive integer from 1 to 10.

110. The fusion protein according to any one of claims 101-109, wherein the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain comprise an amino acid sequence selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (3) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

18. (4) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:17, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (6) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17; (7) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:27, HCDR2 is SEQ ID NO:20, and HCDR3 is SEQ ID NO:18; (8) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:32, HCDR2 is SEQ ID NO:23, and HCDR3 is SEQ ID NO:15; (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18; (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (13) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (14) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (15) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18; and (16) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:

15.

111. The fusion protein according to any one of claims 110-119, wherein the first antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, the second antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain comprise an amino acid sequence selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (3) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (4) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (6) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (7) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (8) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (13) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (14) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (15) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (16) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (17) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (18) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (19) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (20) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

2. (21) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:

2. (22) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (23) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (24) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (25) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (26) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (27) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (28) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (29) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

13. (30) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (31) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (32) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (33) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:

2. (34) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (35) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; and (36) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:

15.

112. The fusion protein according to claims 100-111, wherein the transmembrane region comprises a transmembrane domain derived from one or more proteins selected from the group consisting of: low-density lipoprotein receptor-associated protein or fragments thereof, CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

113. The fusion protein of claim 112, wherein the transmembrane region is a transmembrane region derived from CD8 or a low-density lipoprotein receptor-associated protein or a fragment thereof.

114. The fusion protein of claim 112, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments.

115. The fusion protein according to any one of claims 112-114, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

116. The fusion protein according to any one of claims 100-115, further comprising a co-stimulatory domain, the co-stimulatory signaling region comprising an intracellular co-stimulatory signaling region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

117. The fusion protein of claim 116, wherein the co-stimulatory signal region is an intracellular co-stimulatory signal region derived from 4-1BB or CD28.

118. The fusion protein according to any one of claims 100-117, which binds to the chimeric antigen receptor according to any one of claims 19-43, and / or the chimeric antigen receptor according to any one of claims 44-99.

119. The fusion protein according to any one of claims 100-118, wherein it binds to a chimeric antigen receptor targeting GPRC5D.

120. The fusion protein of claim 119, wherein the chimeric antigen receptor targeting GPRC5D comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises at least one antigen-binding domain.

121. The fusion protein of claim 120, wherein the antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75; (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:

81.

122. The fusion protein according to any one of claims 120-121, wherein the extracellular domain of the chimeric antigen receptor targeting GPRC5D has an antigen-binding domain.

123. The fusion protein according to any one of claims 120-121, wherein the extracellular domain of the chimeric antigen receptor targeting GPRC5D has two antigen-binding domains, a first antigen-binding domain and a second antigen-binding domain.

124. The fusion protein of claim 123, wherein the first antigen-binding domain and the second antigen-binding domain target the same epitope of GPRC5D.

125. The fusion protein of claim 123, wherein the first antigen-binding domain and the second antigen-binding domain target different epitopes of GPRC5D.

126. The fusion protein according to any one of claims 123-125, wherein the first antigen-binding domain and the second antigen-binding domain comprise the same or different amino acid sequences.

127. The fusion protein according to any one of claims 123-126, wherein the first antigen-binding domain and the second antigen-binding domain are directly or indirectly linked.

128. The fusion protein according to any one of claims 123-127, wherein the first antigen-binding domain and the second antigen-binding domain are linked by a linker.

129. The fusion protein according to claim 128, wherein the linker is a linking peptide.

130. The fusion protein according to any one of claims 129, wherein the linker peptide comprises the amino acid sequence (GGGGS)n, wherein n is any positive integer from 1 to 10.

131. The fusion protein according to any one of claims 123-130, wherein the first antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D comprise an amino acid sequence selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:

78. (3) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; (4) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:

78. (6) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; (7) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (8) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81, and the HCDR1 of the second antigen-binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; and (9) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:

81.

132. The fusion protein according to any one of claims 120-121, wherein the extracellular domain of the chimeric antigen receptor targeting GPRC5D has three antigen-binding domains.

133. The fusion protein according to any one of claims 100-132, wherein it binds to a chimeric antigen receptor targeting CD38.

134. The fusion protein of claim 133, wherein the chimeric antigen receptor targeting CD38 comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the extracellular domain comprises at least one antigen-binding domain.

135. The fusion protein according to any one of claims 133-134, wherein the antigen-binding domain targeting CD38 comprises at least one CDR in the variable region VH of the antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112 or SEQ ID NO:

110.

136. The fusion protein according to any one of claims 133-135, wherein the antigen-binding domain targeting CD38 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

102.

137. The fusion protein according to any one of claims 133-136, wherein the antigen-binding domain targeting CD38 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:90 or SEQ ID NO:

93.

138. The fusion protein according to any one of claims 133-137, wherein the antigen-binding domain targeting CD38 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:96 or SEQ ID NO:

100.

139. The fusion protein according to any one of claims 133-138, wherein the antigen-binding domain targeting CD38 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99 or SEQ ID NO:

97.

140. The fusion protein according to any one of claims 133-139, wherein the antigen-binding domain targeting CD38 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:101 or SEQ ID NO:

88.

141. The fusion protein according to any one of claims 133-140, wherein the antigen-binding domain targeting CD38 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:98 or SEQ ID NO:

94.

142. The fusion protein according to any one of claims 133-141, wherein the CD38-targeting antigen-binding domain comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90; (2) HCDR1: SEQ ID NO:101, HCDR2: SEQ ID NO:100, and HCDR3: SEQ ID NO:102; and (3) HCDR1: SEQ ID NO: 101, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO:

102.

143. The fusion protein according to claims 133-142, wherein the antigen-binding domain targeting CD38 comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90; (2) HCDR1: SEQ ID NO:91, HCDR2: SEQ ID NO:92, and HCDR3: SEQ ID NO:93; (3) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:95, and HCDR3: SEQ ID NO:93; (4) HCDR1: SEQ ID NO:98, HCDR2: SEQ ID NO:99, and HCDR3: SEQ ID NO:93; and (5) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:97, and HCDR3: SEQ ID NO:

93.

144. The fusion protein according to any one of claims 120-143, wherein the transmembrane domain comprises a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

145. The fusion protein of claim 144, wherein the transmembrane region is a transmembrane region derived from CD8.

146. The fusion protein according to any one of claims 120-145, wherein the intracellular domain comprises an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

147. The fusion protein of claim 146, wherein the intracellular signaling region is a signal transduction domain derived from CD3ζ.

148. The fusion protein according to any one of claims 118-147, wherein the binding is covalent.

149. The fusion protein of claim 148, wherein the covalent binding is via disulfide bonds.

150. Cells containing and / or expressing a chimeric antigen receptor or fusion protein that targets B cell maturation antigen (BCMA), a chimeric antigen receptor that targets GPRC5D, and / or a fusion protein that targets CD38.

151. The cell of claim 150, wherein the chimeric antigen receptor or fusion protein targeting BCMA comprises at least one antigen-binding domain targeting BCMA.

152. The cell according to any one of claims 150-151, wherein the antigen-binding domain targeting BCMA comprises the antigen-binding protein according to any one of claims 1-18.

153. The cell according to any one of claims 150-152, wherein the chimeric antigen receptor or fusion protein targeting BCMA comprises a BCMA-targeting antigen-binding domain.

154. The cell according to any one of claims 150-152, wherein the chimeric antigen receptor or fusion protein targeting BCMA comprises two BCMA-targeting antigen-binding domains, a first antigen-binding domain targeting BCMA and a second antigen-binding domain targeting BCMA.

155. The cell of claim 154, wherein the first antigen-binding domain targeting BCMA comprises HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting BCMA comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting BCMA and HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting BCMA are each independently selected from any one of the following amino acid sequences: (1) HCDR1: SEQ ID NO:31, HCDR2: SEQ ID NO:24, and HCDR3: SEQ ID NO:16; (2) HCDR1: SEQ ID NO:28, HCDR2: SEQ ID NO:25, and HCDR3: SEQ ID NO:17; (3) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:38, HCDR2: SEQ ID NO:29, HCDR3: SEQ ID NO:20, and HCDR3: SEQ ID NO:18; and (4) HCDR1: SEQ ID NO:32, HCDR2: SEQ ID NO:39, HCDR2: SEQ ID NO:20, HCDR3: SEQ ID NO:20, HCDR3: SEQ ID NO:20, HCDR3: SEQ ID NO:20, HCDR2 ...2: SEQ ID NO:20, HCDR2: SEQ ID NO: NO:23, and HCDR3: SEQ ID NO:

15.

156. The cell according to any one of claims 154-155, wherein the first antigen-binding domain targeting BCMA comprises HCDR1, HCDR2, and HCDR3, the second antigen-binding domain targeting BCMA comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting BCMA and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting BCMA comprise an amino acid sequence selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (3) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

18. (4) The first antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:16, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:17, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (6) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17; (7) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:27, HCDR2 is SEQ ID NO:20, and HCDR3 is SEQ ID NO:18; (8) The first antigen binding domain HCDR1 is SEQ ID NO:28, HCDR2 is SEQ ID NO:25, and HCDR3 is SEQ ID NO:17, and the second antigen binding domain HCDR1 is SEQ ID NO:32, HCDR2 is SEQ ID NO:23, and HCDR3 is SEQ ID NO:15; (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18; (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18, and the second antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (13) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:31, HCDR2 as SEQ ID NO:24, and HCDR3 as SEQ ID NO:

16. (14) The first antigen binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:25, and HCDR3 as SEQ ID NO:

17. (15) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:18; and (16) The first antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:32, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:

15.

157. The cell according to any one of claims 154-156, wherein the first antigen-binding domain targeting BCMA comprises HCDR1, HCDR2, and HCDR3, the second antigen-binding domain targeting BCMA comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting BCMA and the HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting BCMA comprise an amino acid sequence selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (3) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (4) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (6) The first antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (7) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (8) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (9) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (10) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (11) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (12) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (13) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (14) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (15) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (16) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (17) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (18) The first antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (19) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (20) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

2. (21) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:

2. (22) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (23) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; (24) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (25) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (26) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (27) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:2; (28) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (29) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:

13. (30) The first antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13, and the second antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15; (31) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:26, HCDR2 as SEQ ID NO:19, and HCDR3 as SEQ ID NO:1; (32) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:2; (33) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:28, HCDR2 as SEQ ID NO:21, and HCDR3 as SEQ ID NO:

2. (34) The first antigen binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:11; (35) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:27, HCDR2 as SEQ ID NO:20, and HCDR3 as SEQ ID NO:13; and (36) The first antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:15, and the second antigen-binding domain has HCDR1 as SEQ ID NO:29, HCDR2 as SEQ ID NO:23, and HCDR3 as SEQ ID NO:

15.

158. The cell according to any one of claims 150-157, wherein the chimeric antigen receptor targeting GPRC5D comprises at least one antigen-binding domain targeting GPRC5D.

159. The cell of claim 158, wherein the antigen-binding domain targeting GPRC5D comprises at least one CDR in the variable region VH of the antibody heavy chain, the VH comprising SEQ ID NO:83, SEQ ID NO:85 or SEQ ID NO:

87.

160. The cell according to any one of claims 158-159, wherein the antigen-binding domain targeting GPRC5D comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:75, SEQ ID NO:78, or SEQ ID NO:

81.

161. The cell according to any one of claims 158-160, wherein the antigen-binding domain targeting GPRC5D comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:74, SEQ ID NO:77, or SEQ ID NO:

80.

162. The cell according to any one of claims 158-161, wherein the antigen-binding domain targeting GPRC5D comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:73, SEQ ID NO:76, or SEQ ID NO:

79.

163. The cell according to any one of claims 158-162, wherein the antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:73, HCDR2: SEQ ID NO:74, and HCDR3: SEQ ID NO:75; (2) HCDR1: SEQ ID NO:76, HCDR2: SEQ ID NO:77, and HCDR3: SEQ ID NO:78; and (3) HCDR1: SEQ ID NO:79, HCDR2: SEQ ID NO:80, and HCDR3: SEQ ID NO:

81.

164. The cell according to any one of claims 158-163, wherein the antigen-binding domain targeting GPRC5D comprises an antigen-binding domain targeting GPRC5D.

165. The cell according to any one of claims 158-163, wherein the antigen-binding domain targeting GPRC5D comprises two antigen-binding domains targeting GPRC5D, a first antigen-binding domain targeting GPRC5D and a second antigen-binding domain targeting GPRC5D.

166. The cell of claim 165, wherein the first antigen-binding domain and the second antigen-binding domain target the same epitope of GPRC5D.

167. The cell of claim 165, wherein the first antigen-binding domain and the second antigen-binding domain target different epitopes of GPRC5D.

168. The cell according to any one of claims 165-167, wherein the first antigen-binding domain and the second antigen-binding domain comprise the same or different amino acid sequences.

169. The cell according to any one of claims 165-168, wherein the first antigen-binding domain and the second antigen-binding domain are directly or indirectly connected.

170. The cell according to claims 165-169, wherein the first antigen-binding domain and the second antigen-binding domain are connected by a linker.

171. The cell according to claim 170, wherein the linker is a linker peptide.

172. The cell of claim 171, wherein the linker peptide comprises the amino acid sequence (GGGGS)n, wherein n is any positive integer from 1 to 10.

173. The cell according to claim 171, wherein the linker peptide comprises the amino acid sequence (EAAAK)n, wherein n is any positive integer from 1 to 10.

174. The cell according to claims 165-173, wherein the first antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, and the second antigen-binding domain targeting GPRC5D comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 of the first antigen-binding domain targeting GPRC5D and HCDR1, HCDR2, and HCDR3 of the second antigen-binding domain targeting GPRC5D comprise amino acid sequences selected from the group consisting of: (1) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (2) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:

78. (3) The first antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; (4) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (5) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:

78. (6) The first antigen binding domain has HCDR1 as SEQ ID NO:76, HCDR2 as SEQ ID NO:77, and HCDR3 as SEQ ID NO:78, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81; (7) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:73, HCDR2 as SEQ ID NO:74, and HCDR3 as SEQ ID NO:75; (8) The HCDR1 of the first antigen-binding domain is SEQ ID NO:79, the HCDR2 is SEQ ID NO:80, and the HCDR3 is SEQ ID NO:81, and the HCDR1 of the second antigen-binding domain is SEQ ID NO:76, the HCDR2 is SEQ ID NO:77, and the HCDR3 is SEQ ID NO:78; and (9) The first antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:81, and the second antigen binding domain has HCDR1 as SEQ ID NO:79, HCDR2 as SEQ ID NO:80, and HCDR3 as SEQ ID NO:

81.

175. The cell according to any one of claims 158-163, wherein the antigen-binding domain targeting GPRC5D comprises three antigen-binding domains targeting GPRC5D.

176. The cell according to any one of claims 150-175, wherein the antigen-binding domain targeting GPRC5D is directly or indirectly connected to the antigen-binding domain targeting BCMA.

177. The cell according to any one of claims 150-176, wherein the antigen-binding domain targeting GPRC5D and the antigen-binding domain targeting BCMA are connected by a linker.

178. The cell of claim 177, wherein the linker comprises a linker peptide.

179. The cell of claim 178, wherein the linker peptide comprises the amino acid sequence (GGGGS)n, wherein n is any positive integer from 1 to 10.

180. The cell of claim 178, wherein the linker peptide comprises the amino acid sequence (EAAAK)n, wherein n is any positive integer from 1 to 10.

181. The cell according to any one of claims 150-180, wherein the fusion protein targeting BCMA binds to the chimeric antigen receptor targeting GPRC5D.

182. The cell according to any one of claims 150-181, wherein the fusion protein targeting BCMA and the chimeric antigen receptor targeting GPRC5D are covalently bound.

183. The cell according to any one of claims 150-182, wherein the fusion protein targeting BCMA and the chimeric antigen receptor targeting GPRC5D are bound by disulfide bonds.

184. The cell according to any one of claims 150-183, wherein the CD38-targeting fusion protein comprises at least one antigen-binding domain targeting CD38.

185. The cell of claim 184, wherein the antigen-binding domain targeting CD38 comprises at least one CDR in the variable region VH of the antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:112 or SEQ ID NO:

110.

186. The cell according to any one of claims 184-185, wherein the antigen-binding domain targeting CD38 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:

102.

187. The cell according to any one of claims 184-186, wherein the antigen-binding domain targeting CD38 comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:90 or SEQ ID NO:

93.

188. The cell according to any one of claims 184-187, wherein the antigen-binding domain targeting CD38 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:96 or SEQ ID NO:

100.

189. The cell according to any one of claims 184-188, wherein the antigen-binding domain targeting CD38 comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:99 or SEQ ID NO:

97.

190. The cell according to any one of claims 184-189, wherein the antigen-binding domain targeting CD38 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:101 or SEQ ID NO:

88.

191. The cell according to any one of claims 184-190, wherein the antigen-binding domain targeting CD38 comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:98 or SEQ ID NO:

94.

192. The cell according to any one of claims 184-191, wherein the antigen-binding domain targeting CD38 comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90; (2) HCDR1: SEQ ID NO:101, HCDR2: SEQ ID NO:100, and HCDR3: SEQ ID NO:102; and (3) HCDR1: SEQ ID NO: 101, HCDR2: SEQ ID NO: 96, and HCDR3: SEQ ID NO:

102.

193. The cell according to any one of claims 184-192, wherein the antigen-binding domain targeting CD38 comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1-3 comprises an amino acid sequence selected from the group consisting of: (1) HCDR1: SEQ ID NO:88, HCDR2: SEQ ID NO:89, and HCDR3: SEQ ID NO:90; (2) HCDR1: SEQ ID NO:91, HCDR2: SEQ ID NO:92, and HCDR3: SEQ ID NO:93; (3) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:95, and HCDR3: SEQ ID NO:93; (4) HCDR1: SEQ ID NO:98, HCDR2: SEQ ID NO:99, and HCDR3: SEQ ID NO:93; and (5) HCDR1: SEQ ID NO:94, HCDR2: SEQ ID NO:97, and HCDR3: SEQ ID NO:

93.

194. The cell according to any one of claims 150-193, further comprising a transmembrane region comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: low-density lipoprotein receptor-associated protein or fragments thereof, CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

195. The cell of claim 194, wherein the transmembrane region is a transmembrane region derived from CD8, CD28, or low-density lipoprotein receptor-associated protein or a fragment thereof.

196. The cell according to any one of claims 194-195, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more selected from the group consisting of low-density lipoprotein receptor-associated proteins 1-12 and functional fragments thereof.

197. The cell of claim 196, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

198. The cell according to any one of claims 150-197, further comprising a co-stimulatory domain, said co-stimulatory signaling region comprising an intracellular co-stimulatory signaling region derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

199. The cell of claim 198, wherein the co-stimulatory signal region is an intracellular co-stimulatory signal region derived from 4-1BB or CD28.

200. The cell according to any one of claims 150-199, further comprising an intracellular domain comprising an intracellular signaling region derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

201. The cell of claim 200, wherein the intracellular domain is an intracellular signaling region derived from CD3ζ.

202. The cell according to any one of claims 150-201, further comprising a hinge region, said hinge region comprising a hinge region of one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

203. The cell of claim 202, wherein the hinge region is a hinge region derived from CD8 or CD28.

204. The cell according to any one of claims 150-203, further comprising a signal peptide.

205. The cell according to any one of claims 150-204, comprising immune effector cells.

206. The cell according to any one of claims 150-205, comprising T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells and / or kinetic stem cells, or variants thereof.

207. The cell according to claim 206, wherein it is a T cell.

208. An isolated nucleic acid molecule encoding an antigen-binding protein according to any one of claims 1-18, a chimeric antigen receptor according to any one of claims 19-43, a chimeric antigen receptor according to any one of claims 44-99, a fusion protein according to any one of claims 100-149, or a cell according to any one of claims 150-207.

209. The nucleic acid molecule of any one of claims 208, wherein the nucleic acid sequence encoding a chimeric antigen receptor targeting BCMA includes a nucleic acid sequence encoding an antigen-binding domain targeting BCMA.

210. The nucleic acid molecule according to any one of claims 208-209, wherein the antigen-binding domain targeting BCMA comprises the antigen-binding protein according to any one of claims 1-18.

211. The nucleic acid molecule according to any one of claims 208-210, wherein the nucleic acid sequence encoding a chimeric antigen receptor targeting GPRC5D includes a nucleic acid sequence encoding an antigen-binding domain targeting GPRC5D.

212. The nucleic acid molecule according to any one of claims 211, wherein the antigen-binding domain targeting GPRC5D comprises at least one CDR in VH, and the nucleic acid sequence encoding said VH comprises the nucleotide sequence shown in SEQ ID NO:82, SEQ ID NO:84 or SEQ ID NO:

86.

213. The nucleic acid molecule according to any one of claims 211-212, wherein the antigen-binding domain targeting GPRC5D comprises at least one antigen-binding domain targeting GPRC5D.

214. The nucleic acid molecule of claim 213, wherein the antigen-binding domain targeting GPRC5D comprises an antigen-binding domain targeting GPRC5D.

215. The nucleic acid molecule of claim 213, wherein the antigen-binding domain targeting GPRC5D comprises two antigen-binding domains targeting GPRC5D.

216. The nucleic acid molecule according to any one of claims 213, wherein the antigen-binding domain targeting GPRC5D comprises three antigen-binding domains targeting GPRC5D.

217. The nucleic acid molecule according to any one of claims 213-216, wherein the antigen-binding domains targeting GPRC5D are each independently selected from the nucleotide sequences shown in the group consisting of: SEQ ID NO:82, SEQ ID NO:84 and SEQ ID NO:

86.

218. The nucleic acid molecule according to any one of claims 208-217, wherein the nucleic acid sequence encoding the fusion protein targeting CD38 includes a nucleic acid sequence encoding an antigen-binding domain targeting CD38.

219. The nucleic acid molecule of claim 218, wherein the antigen-binding domain targeting CD38 comprises at least one CDR in VH, and the nucleic acid sequence encoding said VH comprises the nucleotide sequence shown in SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:111 or SEQ ID NO:

109.

220. The nucleic acid molecule according to any one of claims 218-219, wherein the antigen-binding domain encoding CD38 comprises the nucleotide sequence shown in SEQ ID NO:103, SEQ ID NO:105 or SEQ ID NO:

109.

221. The nucleic acid molecule according to any one of claims 208-220, comprising a nucleic acid sequence encoding the antigen-binding domain targeting BCMA, a nucleic acid sequence encoding the antigen-binding domain targeting GPRC5D, and / or a nucleic acid sequence encoding the antigen-binding domain targeting CD38.

222. The nucleic acid molecule according to any one of claims 208-221, further comprising a nucleic acid sequence encoding a co-stimulatory signaling domain, said co-stimulatory signaling domain comprising an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the group consisting of: CD28, 4-1BB, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40, and MyD88.

223. The nucleic acid molecule according to any one of claims 208-222, further comprising a nucleic acid sequence encoding an intracellular domain comprising an intracellular domain derived from one or more proteins selected from the group consisting of: CD3ζ, CD3δ, CD3γ, CD3ε, CD79a, CD79b, FcεRIγ, FcεRIβ, FcγRIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14 Nef, Kaposi's sarcoma herpesvirus (HSKV), DAP10, DAP-12, and a domain comprising at least one ITAM.

224. The nucleic acid molecule according to any one of claims 208-223, further comprising a nucleic acid sequence encoding a transmembrane domain, said transmembrane domain comprising a transmembrane domain derived from one or more proteins selected from the group consisting of: CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3ε, CD3ζ, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

225. The nucleic acid molecule according to any one of claims 208-224, further comprising a nucleic acid sequence encoding a hinge region, said hinge region comprising a hinge region derived from one or more proteins selected from the group consisting of: CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

226. The nucleic acid molecule according to any one of claims 208-225, further comprising a nucleic acid sequence encoding a cleavage peptide, said cleavage peptide being selected from 2A peptides.

227. The nucleic acid molecule according to claim 226, wherein the 2A peptide is selected from the group consisting of P2A, T2A, F2A and E2A.

228. The nucleic acid molecule according to any one of claims 208-227, further comprising a nucleic acid sequence encoding a linker.

229. The nucleic acid molecule according to claim 228, wherein the linker is a linker peptide.

230. The nucleic acid molecule of claim 229, wherein the linker peptide comprises an amino acid sequence of (GGGGS)n, wherein n is any positive integer from 1 to 10.

231. The nucleic acid molecule of claim 229, wherein the linker peptide comprises an amino acid sequence of (EAAAK)n, wherein n is any positive integer from 1 to 10.

232. The nucleic acid molecule according to any one of claims 208-231, further comprising a nucleic acid sequence encoding a low-density lipoprotein receptor-associated protein or a fragment thereof.

233. The nucleic acid molecule of claim 232, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof comprises one or more of the following: low-density lipoprotein receptor-associated proteins 1-12 and their functional fragments.

234. The nucleic acid molecule according to any one of claims 232-233, wherein the low-density lipoprotein receptor-associated protein or a fragment thereof is low-density lipoprotein receptor-associated protein 5 and / or 6 or a fragment thereof.

235. The nucleic acid molecule according to any one of claims 208-234, further comprising a nucleic acid sequence encoding a signal peptide, said signal peptide being derived from a CD8 signal peptide.

236. The nucleic acid molecule according to any one of claims 208-235, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the BCMA antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

237. The nucleic acid molecule according to any one of claims 208-236, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, the nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

238. The nucleic acid molecule according to any one of claims 208-237, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the linker, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

239. The nucleic acid molecule according to any one of claims 208-238, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the linker, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

240. The nucleic acid molecule according to any one of claims 208-239, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the linker, the nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the linker, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

241. The nucleic acid molecule according to any one of claims 208-240, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, the nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the hinge region, and a nucleic acid sequence encoding the transmembrane domain.

242. The nucleic acid molecule according to any one of claims 208-241, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, the nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, and a nucleic acid sequence encoding the co-stimulatory signal domain.

243. The nucleic acid molecule according to any one of claims 208-242, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the transmembrane domain, and a nucleic acid sequence encoding the co-stimulatory signal domain.

244. The nucleic acid molecule according to any one of claims 208-243, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, and a nucleic acid sequence encoding the co-stimulatory signal domain.

245. The nucleic acid molecule according to any one of claims 208-244, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target GPRC5D first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the cleavage peptide, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

246. The nucleic acid molecule according to any one of claims 208-245, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target GPRC5D first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the hinge region, and a nucleic acid sequence encoding the transmembrane domain.

247. The nucleic acid molecule according to any one of claims 208-246, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target GPRC5D first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the cleavage peptide, a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target CD38 antigen-binding domain, a nucleic acid sequence encoding the hinge region, and a nucleic acid sequence encoding the transmembrane domain.

248. The nucleic acid molecule according to any one of claims 208-247, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the linker, the nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

249. The nucleic acid molecule according to any one of claims 208-248, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

250. The nucleic acid molecule according to any one of claims 208-249, comprising, from the 5' end to the 3' end, respectively: a nucleic acid sequence encoding the signal peptide, a nucleic acid sequence encoding the target BCMA first antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target GPRC5D antigen-binding domain, a nucleic acid sequence encoding the linker, a nucleic acid sequence encoding the target BCMA second antigen-binding domain, a nucleic acid sequence encoding the hinge region, a nucleic acid sequence encoding the transmembrane domain, a nucleic acid sequence encoding the co-stimulatory signal domain, a nucleic acid sequence encoding the intracellular domain, a nucleic acid sequence encoding the cleavage peptide, and a nucleic acid sequence encoding the low-density lipoprotein receptor-associated protein or a fragment thereof.

251. A vector comprising the nucleic acid molecule of any one of claims 208-250.

252. A method for preparing modified immune cells, comprising introducing the nucleic acid molecule as described in claims 208-250 and / or the vector as described in claim 251 into immune effector cells.

253. The method according to claim 252, wherein the immune cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoprogenitor cells and / or pluripotent stem cells.

254. The method according to any one of claims 252-253, wherein the immune cell is a T cell.

255. A pharmaceutical composition comprising an antigen-binding protein according to any one of claims 1-18, a chimeric antigen receptor according to any one of claims 19-43, a chimeric antigen receptor according to any one of claims 44-99, a fusion protein according to claims 100-149, a nucleic acid molecule according to any one of claims 208-250, a vector according to claim 251, and / or a cell according to any one of claims 150-207, and optionally a pharmaceutically acceptable vector.

256. The antigen-binding protein of any one of claims 1-18, the chimeric antigen receptor of any one of claims 19-43, the chimeric antigen receptor of any one of claims 44-99, the fusion protein of claims 100-149, the cell of any one of claims 150-207, the nucleic acid molecule of any one of claims 208-250, the carrier of claim 251, and / or the pharmaceutical composition of claim 255, for use in the preparation of a medicament for the prevention, treatment and / or relief of diseases or conditions associated with BCMA, GPRC5D and / or CD38 expression.

257. The use according to claim 256, wherein the diseases and / or conditions associated with BCMA, GPRC5D and / or CD38 expression include tumors and / or autoimmune diseases.

258. The use according to any one of claims 256-257, wherein the disease and / or condition includes a solid tumor.

259. The use according to any one of claims 256-257, wherein the disease and / or condition includes non-solid tumors.

260. The use according to any one of claims 256-259, wherein the disease and / or condition includes hematologic malignancies and / or lymphomas.

261. The use according to any one of claims 256-259, wherein the disease and / or condition includes myeloma.

262. A method for preventing, treating, and / or alleviating diseases and / or symptoms associated with BCMA, GPRC5D, and / or CD38 expression, the method comprising administering to a subject in need an antigen-binding protein of any one of claims 1-18, a chimeric antigen receptor of any one of claims 19-43, a chimeric antigen receptor of any one of claims 44-99, a fusion protein of any one of claims 100-149, a cell of any one of claims 150-207, a nucleic acid molecule of any one of claims 208-250, a carrier of any one of claims 251, and / or a pharmaceutical composition of any one of claims 255.

263. The method of claim 262, wherein the diseases and / or conditions associated with BCMA, GPRC5D and / or CD38 expression include tumors and / or autoimmune diseases.

264. The method according to any one of claims 262-263, wherein the disease and / or condition includes a solid tumor.

265. The method according to any one of claims 262-263, wherein the disease and / or condition includes non-solid tumors.

266. The method according to any one of claims 262-265, wherein the disease and / or condition includes hematologic malignancy and / or lymphoma.

267. The method according to any one of claims 262-266, wherein the disease and / or condition includes myeloma.

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