Development and application of novel multi-specific tumor inhibitor

Abstract

Relates to an antibody containing a binding GPRC5D structural domain and application thereof. The GPRC5D * CD3 bispecific antibody has relatively strong killing effect on low expression cells, and meanwhile, the activation on T cells is milder; the GPRC5D * BCMA * CD3 trispecific antibody can kill BCMA + cells and GPRC5D + cells at the same time, is helpful for overcoming drug resistance generated by a single target spot, and can generate a deeper and more lasting tumor inhibition curative effect in clinical application.

Description

Development and application of a new multispecific tumor inhibitor Technical Field

[0001] The present disclosure relates to an antibody containing a GPRC5D binding domain and applications thereof, in particular to a multispecific antibody containing a GPRC5D binding domain and applications thereof.

[0002] background

[0003] Although new therapies are constantly being approved and are revolutionizing the treatment of multiple myeloma, such as the ADC therapy Blenrep (belantamab mafodotin) targeting B-cell maturation antigen (BCMA) approved by the FDA in 2020, the BCMA-targeted CAR-T therapy Abecma (idecabtagene vicleucel) approved in 2021, and the recently approved BCMA-targeted CAR-T therapy Carvykti (ciltacabtagene autoleucel), multiple myeloma is still considered an incurable disease and most patients will eventually relapse. The loss or low expression of BCMA antigen during treatment may lead to tumor recurrence or drug resistance (van de Donk et al., CAR T-cell therapy for multiple myeloma: state of the art and prospects, Lancet Haematol. 2021 Jun; 8(6): e446-e461; Gazeau N et al., Effective anti-BCMA retreatment in multiple myeloma, Blood Adv (2021) 5(15); Da Vià MC et al., Homozygous BCMA gene deletion in response to anti-BCMA CAR T cells in a patient with multiple myeloma, Nat Med. 2021 Apr; 27(4)). Therefore, the development of other new target therapies that do not depend on BCMA, or combination therapies based on different targets, is a reasonable strategy to reduce the recurrence or drug resistance of multiple myeloma caused by antigen loss.

[0004] GPRC5D (G-protein–coupled receptor family C group 5 member D) is an orphan G protein-coupled receptor with a seven-transmembrane structure (Brauner-Osborne H et al., Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D, Biochim Biophys Acta. 2001 Apr 16; 1518(3)). This gene is only expressed in cells that produce hard keratin in the hair follicle part of the skin (Inoue S et al., The RAIG family member, GPRC5D, is associated with hard-keratinized structures, J Invest Dermatol. 2004 Mar; 122(3)). In the analysis of gene expression in myeloma patients, it was found that GPRC5D was specifically highly expressed in multiple myeloma cells and was associated with poor prognosis (Atamaniuk J et al., Overexpression of G protein-coupled receptor 5D in the bone marrow is associated with poor prognosis in patients with multiple myeloma, Eur J Clin Invest. 2012 Sep; 42(9); Cohen Y et al., GPRC5D is a promising marker for monitoring the tumor load and to target multiple myeloma cells, Hematology. 2013 Nov; 18(6)). Smith EL et al. further confirmed that GPRC5D receptor protein is selectively highly expressed in malignant bone marrow plasmacytoma, independent of BCMA expression, and is a new immunotherapy target (Smith EL et al., GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells, Sci Transl Med. 2019 Mar 27; 11(485)).

[0005] Currently, bispecific antibodies and CAR-T therapies targeting GPRC5D have shown significant therapeutic effects on multiple myeloma in preclinical studies.

[0006] Talquetamab (JNJ-64407564) is a GPRC5D×CD3 bispecific antibody developed by Janssen. Preclinical studies have shown that it can mediate effective T cell killing of GPRC5D. + Myeloma cells, the killing effect is significantly positively correlated with the cell GPRC5D expression level (Pillarisetti K et al., A T-cell-redirecting bispecific G-protein-coupled receptor class 5member D×CD3 antibody to treat multiple myeloma, Blood. 2020 Apr 9; 135(15); Verkleij CPM et al., Preclinical activity and determinants of response of the GPRC5D×CD3 bispecific antibody Talquetamab in multiple myeloma, Blood Adv. 2021 Apr 27; 5(8)).

[0007] Kodama T et al. also reported a new GPRC5D×CD3 bispecific antibody, but because it does not recognize the cynomolgus monkey GPRC5D receptor, alternative methods are needed to evaluate preclinical safety (Kodama T et al., Anti-GPRC5D / CD3 Bispecific T-Cell-Redirecting Antibody for the Treatment of Multiple Myeloma, Mol Cancer Ther. 2019 Sep; 18(9)).

[0008] Fertig et al., in US20210054094A1, described two anti-GPRC5D antibodies, 5E11 and 5F11, obtained from rat immunization, which bind to human GPRC5D with high affinity. + Stably transfected cells, but does not bind to cynomolgus monkey GPRC5D + Stably transfected cells; although the other two antibodies 10B10 and B72 bound to cynomolgus monkey GPRC5D, the binding level was low and the TDCC activity mediated was weak.

[0009] Fernández de Larrea C et al. reported that CAR-T therapy that simultaneously targets BCMA and GPRC5D can prevent recurrence caused by BCMA escape in a mouse transplant tumor model (Fernández de Larrea C et al., Defining an Optimal Dual-Targeted CAR T-cell Therapy Approach Simultaneously Targeting BCMA and GPRC5D to Prevent BCMA Escape-Driven Relapse in Multiple Myeloma, Blood Cancer Discov. 2020 Sep; 1(2)).

[0010] Although the above-mentioned drugs developed based on GPRC5D-targeted antibodies have shown efficacy in preclinical trials, better treatments are still needed in clinical practice to reduce recurrence and drug resistance caused by antigen escape, and to reduce side effects such as cytokine release syndrome and neurotoxicity produced during treatment.

[0011] Summary of the Invention

[0012] In their research, the inventors used different mouse immunization strategies to discover multiple anti-GPRC5D monoclonal antibodies that can bind to human and cynomolgus macaque GPRC5D receptors on cell membranes with high affinity. They also unexpectedly discovered that a new humanized GPRC5D×CD3 bispecific antibody constructed based on the anti-CD3e antibody SP34 (Pessano et al., The T3 / T cell receptor complex: antigenic distinction between the two 20-kd T3 (T3-delta and T3-epsilon) subunits, EMBO J. 1985 Feb; 4(2)) has strong killing effects on low-expressing cells while being more moderate in activating T cells, thus potentially leading to better clinical efficacy and safety. The inventors also further constructed a GPRC5D×BCMA×CD3 trispecific antibody, which showed superior efficacy to the BCMA×CD3 bispecific antibody in preclinical mouse models and may produce a more sustained response.

[0013] In one aspect, the disclosure provides an antibody, or antigen-binding portion thereof, that binds GPRC5D.

[0014] In another aspect, the present disclosure provides nucleic acids encoding the aforementioned antibodies or antigen-binding portions thereof.

[0015] In another aspect, the present disclosure provides a multispecific antibody, or antigen-binding portion thereof, comprising at least:

[0016] (a) the aforementioned antibody or antigen-binding fragment thereof that binds to the GPRC5D antigen, which is a first antigen-binding portion, wherein the GPRC5D antigen is a first antigen, the first antigen-binding portion comprises a first heavy chain variable region and a first light chain variable region, and the first antigen-binding portion comprises a first binding domain that binds to the first antigen; and

[0017] (b) an antibody or antigen-binding fragment thereof that binds to the CD3 antigen, which is a second antigen-binding portion, wherein the CD3 antigen is a second antigen, the second antigen-binding portion comprises a second heavy chain variable region and a second light chain variable region, and the second antigen-binding portion comprises a second binding domain that binds to the second antigen;

[0018] More preferably, the multispecific antibody or antigen-binding portion thereof further comprises:

[0019] (c) an antibody or antigen-binding fragment thereof that binds to the BCMA antigen, which is a third antigen-binding portion, wherein the BCMA antigen is a third antigen, the third antigen-binding portion comprises a third heavy chain variable region and a third light chain variable region, and the third antigen-binding portion comprises a third binding domain that binds to the third antigen.

[0020] In another aspect, the present disclosure provides nucleic acids encoding the aforementioned multispecific antibodies or antigen-binding portions thereof.

[0021] In another aspect, the present disclosure provides a vector encoding the aforementioned nucleic acid.

[0022] In another aspect, the present disclosure provides a cell comprising the aforementioned vector.

[0023] In another aspect, the present disclosure provides a composition comprising the aforementioned antibody or antigen-binding portion thereof that binds to GPRC5D, the aforementioned multispecific antibody or antigen-binding portion thereof, the aforementioned nucleic acid, and / or the aforementioned cell.

[0024] In another aspect, the present disclosure provides a kit comprising the aforementioned antibody or antigen-binding portion thereof that binds to GPRC5D, the aforementioned multispecific antibody or antigen-binding portion thereof, the aforementioned nucleic acid, the aforementioned cell, and / or the aforementioned composition.

[0025] In another aspect, the present disclosure provides a method for preparing a medicament or kit for diagnosing, treating or preventing cancer or autoimmune diseases comprising the aforementioned antibody or antigen-binding portion thereof that binds to GPRC5D, the aforementioned multispecific antibody or antigen-binding portion thereof, the aforementioned nucleic acid, the aforementioned cell and / or the aforementioned composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG1 shows the flow cytometry detection results of human and cynomolgus monkey GPRC5D HEK293 stable cell lines.

[0027] FIG2 shows the flow cytometry detection results of human and cynomolgus monkey GPRC5D CHO stable cell lines.

[0028] Figure 3 shows the binding of anti-GPRC5D chimeric antibodies to GPRC5D stably transfected cell lines, wherein Figure 3A shows the binding of anti-GPRC5D chimeric antibodies to human GPRC5D stably transfected cell lines; Figure 3B shows the binding of anti-GPRC5D chimeric antibodies to cynomolgus monkey GPRC5D stably transfected cell lines.

[0029] FIG4 shows the binding of the GPRC5D×CD3κλ bispecific antibody to MM.1R tumor cells.

[0030] FIG5 shows that GPRC5D×CD3κλ bispecific antibody mediates T cell killing of MM.1R tumor cells.

[0031] FIG6 shows the binding of the 2F1 GPRC5D×CD3κλ bispecific antibody and its variants to MM.1R tumor cells.

[0032] Figure 7 shows the binding of the 2F1 GPRC5D×CD3κλ bispecific antibody variant to the GPRC5D stably transfected cell line, wherein 7A shows the binding of the 2F1 GPRC5D×CD3κλ bispecific antibody variant to the human GPRC5D stably transfected cell line; Figure 7B shows the binding of the 2F1 GPRC5D×CD3κλ bispecific antibody variant to the cynomolgus monkey GPRC5D stably transfected cell line.

[0033] FIG8 shows binding of 2F1 GPRC5D×CD3κλ bispecific antibody variants to Jurkat cells.

[0034] Figure 9 shows TDCC mediated by 2F1GPRC5D×CD3κλ bispecific antibody variants, wherein Figure 9A shows the TDCC activity of 2F1GPRC5D×CD3κλ bispecific antibody variants in mediating T cell killing of MM.1R tumor cells; Figure 9B shows the process of CD4 + T and CD8 + Changes in the expression of cell surface activation receptors CD69 and CD25.

[0035] FIG10 shows the activation of the T cell NFAT signaling pathway by the 2F1 GPRC5D×CD3κλ bispecific antibody variant.

[0036] FIG11 shows the inhibition of tumor growth by 2F1 GPRC5D×CD3κλ bispecific antibody variants in a mouse xenograft tumor model.

[0037] FIG12 shows the changes in mouse body weight after using 2F1 GPRC5D×CD3κλ bispecific antibody variants in a mouse xenograft tumor model.

[0038] FIG13 shows the GPRC5D×BCMA×CD3 triTCE trispecific antibody structure.

[0039] FIG. 14 shows the interaction between GPRC5D×BCMA×CD3 triTCE antibody and GPRC5D + Combination of stably transfected cells.

[0040] FIG15 shows the interaction between GPRC5D×BCMA×CD3 triTCE antibody and BCMA + Combination of stably transfected cells.

[0041] FIG. 16 shows the binding of GPRC5D×BCMA×CD3 triTCE antibody to Jurkat cells.

[0042] FIG17 shows the effect of GPRC5D×BCMA×CD3 triTCE antibody on GPRC5D + , BCMA + Stable cell lines and GPRC5D + / BCMA + TDCC activity in tumor cells, wherein FIG17A shows the effect of GPRC5D×BCMA×CD3 triTCE antibody on BCMA + Figure 17B shows the killing effect of GPRC5D×BCMA×CD3 triTCE antibody on GPRC5D + Figure 17C shows the killing effect of GPRC5D×BCMA×CD3 triTCE antibody on the myeloma cell line MM.1S that simultaneously expresses both GPRC5D and BCMA receptors; Figure 17D shows the killing effect of GPRC5D×BCMA×CD3 triTCE antibody on the myeloma cell line NCI-H929 that simultaneously expresses both GPRC5D and BCMA receptors. DETAILED DESCRIPTION

[0043] I. Definition

[0044] In this disclosure, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are those widely used in the respective fields and are common procedures. To facilitate a better understanding of this disclosure, definitions and explanations of relevant terms are provided below.

[0045] Provided herein are antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to GPRC5D. In a specific aspect, provided herein are monoclonal anti-GPRC5D antibodies that specifically bind to GPRC5D, wherein the anti-GPRC5D antibodies include variants of parent antibodies. In a specific aspect, provided herein are antibodies that specifically bind to GPRC5D (e.g., human GPRC5D). In a specific aspect, provided herein are anti-GPRC5D antibodies comprising modifications in one or more amino acid residues (e.g., 1-3 amino acid substitutions in the framework region of the heavy chain variable region), which maintain affinity for the antigen compared to the parent antibody without the modifications.

[0046] As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0047] With respect to antibody chain polypeptide sequences, the phrase "substantially identical" is understood to mean antibody chains that exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the phrase is understood to mean nucleotide sequences that exhibit at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

[0048] Sequence "identity" or "identity" has an art-recognized meaning, and published techniques can be used to calculate the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0049] "Substitution" variants are variants in which at least one amino acid residue in the native sequence has been removed and a different amino acid has been inserted in the same position. The substitutions can be single, where only one amino acid is substituted in the molecule, or multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions can be at consecutive sites. Likewise, a single amino acid can be substituted by multiple residues, where such variants include both substitutions and insertions. "Insertion" variants are variants in which one or more amino acids are inserted adjacent to an amino acid at a specific position in a native sequence. Adjacent to an amino acid means linked to the α-carboxyl or α-amino functional group of that amino acid. "Deletion" variants are variants in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of the molecule.

[0050] In the context of antibody variable domains, the term "variable" refers to those portions of the related molecule that exhibit extensive sequence variation between antibodies and are used by a given antibody to specifically recognize and bind its specific target. However, variability is not evenly distributed throughout the variable domain of an antibody. Variability is concentrated in three segments known as the complementarity-determining regions (CDRs; i.e., CDR1, CDR2, and CDR3), or hypervariable regions, located within the variable domains of both the light and heavy chains. The more highly conserved portions of the variable domains are known as the framework (FR) regions or framework sequences. Each variable domain of a native heavy and light chain consists of four FR regions, which primarily adopt a β-sheet configuration and are connected by three CDRs, which form loops that connect and, in some cases, form part of the β-sheet structure. The CDRs of each chain are typically linked adjacently by the FR regions and, with the help of CDRs from the other chain, contribute to the formation of the antibody's target binding site (epitope or determinant). As used herein, the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. A CDR may have the ability to specifically bind to a cognate epitope.

[0051] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains binding specificity and at least part of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was derived. Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, such as recombinantly produced derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may comprise multiple chains linked together, for example, by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, when inserted into an antibody framework (e.g., by replacing the corresponding region), obtains immunospecific binding (i.e., exhibits at least or at least about 10 7 -10 8 "Functional fragments" or "analogs of anti-GPRC5D antibodies" are fragments or analogs that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, functional fragments generally have the same meaning as "antibody fragments" and, with respect to antibodies, can refer to fragments that can prevent or substantially reduce the ability of the receptor to bind to a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. "Fv" fragments are dimers (V H -V L In this configuration, the three CDRs of each variable domain interact to determine the V H -V L The target binding site on the surface of the dimer is the same as that of an intact antibody. The six CDRs collectively confer target binding specificity to the intact antibody. However, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) can still have the ability to recognize and bind to a target.

[0052] As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This characteristic is in contrast to the characteristic of a polyclonal population of antibodies, which comprises antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by many well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalized B cells, for example, by fusion with myeloma cells to produce hybridoma cell lines or by infecting B cells with viruses such as EBV. Recombinant technology can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0053] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and antibodies with the same domains produced synthetically.

[0054] The term "chimeric antibody" refers to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as antibodies in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0055] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.

[0056] In addition, in humanization, it is also possible to mutate the amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL to thereby improve one or more binding properties (e.g., affinity) of the antibody. For example, PCR-mediated mutations can be used to introduce mutations, and their effects on antibody binding or other functional properties can be evaluated using in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. In addition, the mutations in the CDRs are typically no more than one or two. Therefore, the humanized antibodies described herein also encompass antibodies comprising one or two amino acid mutations in the CDRs.

[0057] "Conservative amino acid substitutions" may involve replacing a native amino acid residue with a non-native residue so that the polarity or charge of the amino acid residue at that position is less or has no effect. Examples of conservative substitutions include replacing one non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, norleucine, alanine or methionine with another; replacing one polar (hydrophilic) amino acid residue with another, such as a substitution between arginine and lysine, a substitution between glutamine and asparagine, a substitution between glycine and serine; replacing one basic amino acid residue such as lysine, arginine or histidine with another; or replacing one acidic residue such as aspartic acid or glutamic acid with another. The phrase "conservative amino acid substitutions" also includes replacing a non-derivatized residue with a chemically derivatized residue, provided that the polypeptide exhibits the desired biological activity. Other exemplary amino acid substitutions that can be used according to the present invention are shown in Table 1 below.

[0058] Table 1 Some useful amino acid substitutions.

[0059] As used herein, the term "CDR" refers to a complementarity-determining region, and each heavy chain and light chain of a known antibody molecule has three CDRs. CDRs are also referred to as hypervariable regions and are present in the variable regions of each heavy and light chain of an antibody, with very high variability sites in the primary structure of the CDRs. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0060] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants typically comprise chemically active surface patterns of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.

[0061] As used herein, "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody combining sites of the antibody and the antigen. The antigen may be an isolated antigen or present on a tumor cell. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present at a level of about 1 × 10 7 M -1 or 1×10 8 M -1 or greater affinity constant Ka (or 1×10 -7 M or 1×10 -8 The affinity constant can be determined by standard kinetic methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11: 54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); see also U.S. Patent No. 7,229,619 for exemplary SPR and ITC methods for calculating the binding affinity of antibodies). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, Malmqvist (2000) Biochem. Soc. Trans. 27: 335).

[0062] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0063] Alternatively, in another embodiment, mutations can be introduced randomly along all or a portion of the anti-GPRC5D antibody coding sequence by, for example, saturation mutagenesis, and the resultant modified anti-GPRC5D antibodies can be screened for improved binding activity.

[0064] As used herein, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0065] As used herein, a "host cell" is a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells, such as HEK 293 cells.

[0066] As used herein, a "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the purpose of amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are typically kept episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0067] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as vehicles or shuttles) the exogenous genes into cells.

[0068] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain episomal or that are integrated into the host cell genome.

[0069] As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or progression of the disease. Treatment also includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided, as well as the compositions provided herein.

[0070] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0071] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

[0072] As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0073] As used herein, the term "patient" or "subject" refers to a mammal, such as a human.

[0074] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0075] II. Detailed description of specific implementation methods

[0076] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to the GPRC5D antigen, comprising:

[0077] (1) a heavy chain variable region and a light chain variable region, wherein:

[0078] (i) the heavy chain variable region comprises:

[0079] (a) a HCDR1 sequence having the formula:

[0080] TSYNMH (SEQ ID NO. 5); and

[0081] (b) a HCDR2 sequence having the formula:

[0082] YIYPGNGGTNYNQX1FX2X3 (SEQ ID NO. 242), wherein:

[0083] X1 is K or N, X2 is K or Q, and X3 is A or G; and

[0084] (c) a HCDR3 sequence having the formula:

[0085] GYGYRYWYFDV (SEQ ID NO. 7); and

[0086] (ii) the light chain variable region comprises:

[0087] (a) a LCDR1 sequence having the formula:

[0088] X4ASSSVX5X6MH (SEQ ID NO. 243), wherein:

[0089] X4 is S or R, X5 is S or R, and X6 is Y or F; and

[0090] (b) a LCDR2 sequence having the formula:

[0091] DTSKX7AS (SEQ ID NO. 244), wherein:

[0092] X7 is V or L; and

[0093] (c) a LCDR3 sequence having the formula:

[0094] QQWX8X9X10 PX 11 T (SEQ ID NO. 245), wherein:

[0095] X8 is S or N, X9 is S or N, X 10 is N, Y or K, X 11 is L or P; or

[0096] (2) a heavy chain variable region and a light chain variable region, wherein:

[0097] (i) the heavy chain variable region comprises:

[0098] (a) a HCDR1 sequence having the formula:

[0099] TGYTMN (SEQ ID NO. 25); and

[0100] (b) a HCDR2 sequence having the formula:

[0101] LINPYNGGX 12 X 13 YNX 14 KFX 15 G (SEQ ID NO. 246), wherein:

[0102] X 12 S or T, X 13 R, S or N, X 14 Q or L, X 15 is a King or Queen; and

[0103] (c) a HCDR3 sequence having the formula:

[0104] X 16 X 17 X 18 RX 19 X 20 MDY (SEQ ID NO. 247), wherein:

[0105] X 16 W or L, X 17 A, P or G, X 18 I or L, X 19 Y or N, X 20 is A or G; and

[0106] (ii) the light chain variable region comprises:

[0107] (a) a LCDR1 sequence having the formula:

[0108] X 21 ASQX 22 X 23 X24 X 25 NX 26 X 27 (SEQ ID NO. 248), wherein:

[0109] X 21 is K, R or Q, X 22 N or S, X 23 V or I, X 24 G or S, X 25 T or N, X 26 V or L, X 27 is A or H; and

[0110] (b) a LCDR2 sequence having the formula:

[0111] X 28 ASX 29 X 30 X 31 S (SEQ ID NO. 249), wherein:

[0112] X 28 F, Y or S, X 29 is Y, L or Q, X 30 R, L or S, X 31 is N or 1; and

[0113] (c) a LCDR3 sequence having the formula:

[0114] QQX 32 NSX 33 X 34 X 35 T (SEQ ID NO. 250), wherein:

[0115] X 32 Y or S, X 33 is Y or W, X 34 L or P, X 35 is L or Q; or

[0116] (3) a heavy chain variable region and a light chain variable region, wherein:

[0117] (i) the heavy chain variable region comprises:

[0118] (a) a HCDR1 sequence having the formula:

[0119] TNFPIE (SEQ ID NO. 42); and

[0120] (b) a HCDR2 sequence having the formula:

[0121] NFHPYNDVTKYNEKFX36 G (SEQ ID NO. 251), wherein:

[0122] X 36 is a King or Queen; and

[0123] (c) a HCDR3 sequence having the formula:

[0124] ITRGY (SEQ ID NO. 44); and

[0125] (ii) the light chain variable region comprises:

[0126] (a) a LCDR1 sequence having the formula:

[0127] KSSQSLLYSRNQKNSLA (SEQ ID NO. 47); and

[0128] (b) a LCDR2 sequence having the formula:

[0129] WASTRES (SEQ ID NO. 48); and

[0130] (c) a LCDR3 sequence having the formula:

[0131] QQYYSYPLT (SEQ ID NO. 49); or

[0132] (4) a heavy chain variable region and a light chain variable region, wherein:

[0133] (i) the heavy chain variable region comprises:

[0134] (a) a HCDR1 sequence having the formula:

[0135] TSYX 37 MY (SEQ ID NO. 252), wherein:

[0136] X 37 is Y or T; and

[0137] (b) a HCDR2 sequence having the formula:

[0138] X 38 INPSX 39 GX 40 TX 41 YNX 42 KFX 43 X 44 (SEQ ID NO. 253), wherein:

[0139] X 38 G or Y, X 39 N or S, X 40G or Y, X 41 K or N, X 42 Q or E, X 43 K or Q, X 44 is S, D or G; and

[0140] (c) a HCDR3 sequence having the formula:

[0141] X 45 X 46 X 47 X 48 X 49 X 50 X 51 YX 52 X 53 X 54 X 55 DY (SEQ ID NO. 254), wherein:

[0142] X 45 G or W, X 46 G or S, X 47 G or L, X 48 R or N, X 49 E or S, X 50 Y or R, X 51 Y or L, X 52 A or Y, X 53 Y or M, X 54 A or not present, X 55 is M or does not exist; and

[0143] (ii) the light chain variable region comprises:

[0144] (a) a LCDR1 sequence having the formula:

[0145] X 56 ASX 57 X 58 X 59 X 60 X 61 YX 62 X 63 (SEQ ID NO. 255), wherein:

[0146] X 56 R, Q or S, X 57 Q or S, X 58 D or S, X 59 I or V, X 60 S or not present, X 61 N or S, X 62 For L or M, X 63 is N or Y; and

[0147] (b) a LCDR2 sequence having the formula:

[0148] X 64 TSX 65 LX 66 S (SEQ ID NO. 256), wherein:

[0149] X 64 is Y or G, X 65 R or N, X 66 is H or A; and

[0150] (c) a LCDR3 sequence having the formula:

[0151] QQX 67 X 68 SX 69 PX 70 T (SEQ ID NO. 257), wherein:

[0152] X 67 R or G, X 68 Y or S, X 69 L or Y, X 70 is Y or L; or

[0153] (5) a heavy chain variable region and a light chain variable region, wherein:

[0154] (i) the heavy chain variable region comprises:

[0155] (a) a HCDR1 sequence having the formula:

[0156] TX 71 YVMX 72 (SEQ ID NO. 258), wherein:

[0157] X 71 S or N, X 72 is Y or H; and

[0158] (b) a HCDR2 sequence having the formula:

[0159] YINPYNDGTKYNEX 73 FX 74 G (SEQ ID NO. 259), wherein:

[0160] X 73 K or R, X 74 is King, Ten or Queen; and

[0161] (c) a HCDR3 sequence having the formula:

[0162] GGX75 X 76 X 77 X 78 X 79 X 80 X 81 X 82 X 83 Y (SEQ ID NO. 260), wherein:

[0163] X 75 M or V, X 76 L or R, X 77 T or R, X 78 T or Y, X 79 R or F, X 80 S or not present, X 81 L or not present, X 82 F or not present, X 83 is A, T, V, L, or D; and

[0164] (ii) the light chain variable region comprises:

[0165] (a) a LCDR1 sequence having the formula:

[0166] RASQX 84 IX 85 X 86 X 87 X 88 X 89 (SEQ ID NO. 261), wherein:

[0167] X 84 R, E or D, X 85 G or S, X 86 G or S, X 87 Y or N, X 88 L or F, X 89 is N or S; and

[0168] (b) a LCDR2 sequence having the formula:

[0169] AX 90 SX 91 LDS (SEQ ID NO. 262), wherein:

[0170] X 90 A or T, X 91 is T or S; and

[0171] (c) a LCDR3 sequence having the formula:

[0172] LQX 92 X 93 PX94 T (SEQ ID NO. 263), wherein:

[0173] X 92 S or N, X 93 Y or F, X 94 is P or Y; or

[0174] (6) a heavy chain variable region and a light chain variable region, wherein:

[0175] (i) the heavy chain variable region comprises:

[0176] (a) a HCDR1 sequence having the formula:

[0177] SSYGMS (SEQ ID NO. 58); and

[0178] (b) a HCDR2 sequence having the formula:

[0179] TISSGGSYTYYPDSVKG (SEQ ID NO. 59); and

[0180] (c) a HCDR3 sequence having the formula:

[0181] HX 95 X 96 X 97 X 98 X 99 X 100 X 101 X 102 DY (SEQ ID NO. 264), wherein:

[0182] X 95 G or E, X 96 G or A, X 97 S or R, X 98 S or A, X 99 S or T, X 100 Y or F, X 101 V or not present, X 102 is M or does not exist; and

[0183] (ii) the light chain variable region comprises:

[0184] (a) a LCDR1 sequence having the formula:

[0185] RSSKSLLHSNGNTYLY(SEQ ID NO.63); and

[0186] (b) a LCDR2 sequence having the formula:

[0187] RMSNLAS (SEQ ID NO. 64); and

[0188] (c) a LCDR3 sequence having the formula:

[0189] MQHLEYPFT (SEQ ID NO. 65); or

[0190] (7) a heavy chain variable region and a light chain variable region, wherein:

[0191] (i) the heavy chain variable region comprises:

[0192] (a) a HCDR1 sequence having the formula:

[0193] TRYWMH (SEQ ID NO. 15); and

[0194] (b) a HCDR2 sequence having the formula:

[0195] EFNPSNGRINYNEKFX 103 X 104 (SEQ ID NO. 265), wherein:

[0196] X 103 K or Q, X 104 is N or G; and

[0197] (c) a HCDR3 sequence having the formula:

[0198] GFAY (SEQ ID NO. 17); and

[0199] (ii) the light chain variable region comprises:

[0200] (a) a LCDR1 sequence having the formula:

[0201] X 105 ASSTVSYIH (SEQ ID NO. 266), wherein:

[0202] X 105 is S or R; and

[0203] (b) a LCDR2 sequence having the formula:

[0204] DTSKLAS (SEQ ID NO. 21); and

[0205] (c) a LCDR3 sequence having the formula:

[0206] QQWTTNPWT (SEQ ID NO. 22).

[0207] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D, comprising a heavy chain CDR1 selected from the group consisting of amino acid sequences of SEQ ID NO. 5, 15, 25, 42, 58, 68, 98, 108, 119, 241, or any variant thereof, a heavy chain CDR2 selected from the group consisting of amino acid sequences of SEQ ID NO. 6, 16, 26, 35, 43, 52, 59, 69, 78, 89, 99, 115, 133, 147, or any variant thereof, a heavy chain CDR3 selected from the group consisting of amino acid sequences of SEQ ID NO. 7, 17, 27, 44, 60, 70, 79, 90, 100, 109, 116, 120, 134, 139, 148, or any variant thereof; and a heavy chain CDR4 selected from the group consisting of amino acid sequences of SEQ ID NO. NO.10, 20, 30, 38, 47, 63, 73, 82, 93, 103, 112, 123, 130, 167, 173, 179, 190, 208 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO.11, 21, 31, 48, 64, 74, 83, 94, 104, 124, 151, 209 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO.12, 22, 32, 39, 49, 55, 65, 75, 84, 95, 105, 125, 144 or any variant thereof.

[0208] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D, comprising a heavy chain and light chain CDR combination selected from:

[0209] (1) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 6, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 10, 11, and 12, respectively;

[0210] (2) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 15, 16, and 17, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 20, 21, and 22, respectively;

[0211] (3) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 26, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 30, 31, and 32, respectively;

[0212] (4) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 35, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 38, 21, and 39, respectively;

[0213] (5) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 42, 43, and 44, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 47, 48, and 49, respectively;

[0214] (6) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 52, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 38, 21, and 55, respectively;

[0215] (7) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 58, 59, and 60, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 63, 64, and 65, respectively;

[0216] (8) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 68, 69, and 70, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 73, 74, and 75, respectively;

[0217] (9) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 241, 78, and 79, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 82, 83, and 84, respectively;

[0218] (10) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 42, 43, and 44, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 47, 48, and 49, respectively;

[0219] (11) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 89, and 90, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 93, 94, and 95, respectively;

[0220] (12) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 99, and 100, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 103, 104, and 105, respectively;

[0221] (13) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 108, 99, and 109, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 112, 104, and 105, respectively;

[0222] (14) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 115, and 116, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 112, 104, and 105, respectively;

[0223] (15) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 119, 99, and 120, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 123, 124, and 125, respectively;

[0224] (16) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 108, 99, and 116, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 130, 104, and 105, respectively;

[0225] (17) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 108, 133, and 134, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 112, 104, and 105, respectively;

[0226] (18) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 58, 59 and 139, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 63, 64 and 65, respectively;

[0227] (19) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 99, and 109, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 103, 104, and 105, respectively;

[0228] (20) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 99, and 109, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 103, 104, and 144, respectively;

[0229] (21) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 147, and 148, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 93, 151, and 95, respectively;

[0230] (22) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 15, 164 and 17, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 167, 21 and 22, respectively;

[0231] (23) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 170, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 173, 31, and 32, respectively;

[0232] (24) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 176, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 179, 21, and 39, respectively;

[0233] (25) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 42, 182, and 44, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 47, 48, and 49, respectively;

[0234] (26) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 68, 187, and 70, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 190, 74, and 75, respectively;

[0235] (27) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 119, 193, and 120, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 123, 124, and 125, respectively;

[0236] (28) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 170, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 208, 209, and 32, respectively;

[0237] (29) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 170, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 173, 209, and 32, respectively;

[0238] (30) The heavy chain CDR1, CDR2 and CDR3 sequences comprise SEQ ID NOs. 25, 170 and 27, respectively, and the light chain CDR1, CDR2 and CDR3 sequences comprise SEQ ID NOs. 208, 31 and 32, respectively.

[0239] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that binds to GPRC5D, comprising a heavy chain variable region selected from the group consisting of the amino acid sequence of SEQ ID NO. 3, 13, 23, 33, 40, 50, 56, 66, 76, 87, 96, 106, 113, 117, 126, 131, 140, 145, 162, 168, 174, 180, 185, 191, 202, 232, or any variant thereof, and a heavy chain variable region selected from the group consisting of the amino acid sequence of SEQ ID NO. NO. 8, 18, 28, 36, 45, 53, 61, 71, 80, 85, 91, 101, 110, 121, 128, 135, 142, 149, 165, 171, 177, 183, 188, 194, 206, 212, 216, 220, 230, or any variant thereof.

[0240] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 3 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 8 or any variant thereof.

[0241] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 13 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 18 or any variant thereof.

[0242] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 23 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 28 or any variant thereof.

[0243] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 33 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 36 or any variant thereof.

[0244] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 40 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 45 or any variant thereof.

[0245] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 50 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 53 or any variant thereof.

[0246] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 56 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 61 or any variant thereof.

[0247] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 66 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 71 or any variant thereof.

[0248] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 76 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 80 or any variant thereof.

[0249] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 40 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 85 or any variant thereof.

[0250] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 87 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 91 or any variant thereof.

[0251] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 96 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 101 or any variant thereof.

[0252] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 106 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 110 or any variant thereof.

[0253] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 113 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 110 or any variant thereof.

[0254] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 117 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 121 or any variant thereof.

[0255] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 126 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 128 or any variant thereof.

[0256] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 131 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 135 or any variant thereof.

[0257] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 137 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 61 or any variant thereof.

[0258] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 140 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 101 or any variant thereof.

[0259] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 140 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 142 or any variant thereof.

[0260] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 145 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 149 or any variant thereof.

[0261] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 162 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 165 or any variant thereof.

[0262] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 168 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 171 or any variant thereof.

[0263] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 174 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 177 or any variant thereof.

[0264] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 180 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 183 or any variant thereof.

[0265] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 185 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 188 or any variant thereof.

[0266] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 191 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 194 or any variant thereof.

[0267] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 202 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 206 or any variant thereof.

[0268] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 202 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 212 or any variant thereof.

[0269] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 202 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 216 or any variant thereof.

[0270] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 202 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 220 or any variant thereof.

[0271] In a preferred embodiment, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO. 232 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO. 230 or any variant thereof.

[0272] In a preferred embodiment, the antibody or antigen-binding fragment thereof is humanized.

[0273] In another aspect, the present disclosure provides nucleic acids encoding the aforementioned antibodies or antigen-binding fragments thereof.

[0274] In a preferred embodiment, the nucleic acid comprises an antibody heavy chain variable region nucleic acid sequence selected from the group consisting of SEQ ID NO. 4, 14, 24, 34, 41, 51, 57, 67, 77, 88, 97, 107, 114, 118, 127, 132, 138, 141, 146, 163, 169, 175, 181, 186, 192, 203, or any variant thereof, and an antibody light chain variable region nucleic acid sequence selected from the group consisting of SEQ ID NO. 9, 19, 29, 37, 46, 54, 62, 72, 81, 86, 92, 102, 111, 122, 129, 136, 143, 150, 166, 172, 178, 184, 189, 195, 207, 213, 217, 221, or any variant thereof.

[0275] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 4 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 9 or any variant thereof.

[0276] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 14 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 19 or any variant thereof.

[0277] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 24 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 29 or any variant thereof.

[0278] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 34 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 37 or any variant thereof.

[0279] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 41 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 46 or any variant thereof.

[0280] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 51 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 54 or any variant thereof.

[0281] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 57 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 62 or any variant thereof.

[0282] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 67 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 72 or any variant thereof.

[0283] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 77 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 81 or any variant thereof.

[0284] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 41 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 86 or any variant thereof.

[0285] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 88 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 92 or any variant thereof.

[0286] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 97 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 102 or any variant thereof.

[0287] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 107 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 111 or any variant thereof.

[0288] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 114 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 111 or any variant thereof.

[0289] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 118 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 122 or any variant thereof.

[0290] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 127 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 129 or any variant thereof.

[0291] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 132 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 136 or any variant thereof.

[0292] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 138 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 62 or any variant thereof.

[0293] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 141 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 102 or any variant thereof.

[0294] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 141 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 143 or any variant thereof.

[0295] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 146 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 150 or any variant thereof.

[0296] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 163 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 166 or any variant thereof.

[0297] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 169 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 172 or any variant thereof.

[0298] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 175 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 178 or any variant thereof.

[0299] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 181 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 184 or any variant thereof.

[0300] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 186 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 189 or any variant thereof.

[0301] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 192 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 195 or any variant thereof.

[0302] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 207 or any variant thereof.

[0303] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 213 or any variant thereof.

[0304] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 217 or any variant thereof.

[0305] In a more preferred embodiment, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 221 or any variant thereof.

[0306] In another aspect, the present disclosure provides a multispecific antibody or antigen-binding fragment thereof comprising at least:

[0307] (a) An antibody or antigen-binding fragment thereof that binds to the GPRC5D antigen according to any one of claims 1 to 4, which is a first antigen-binding portion, wherein the GPRC5D antigen is a first antigen, and the first antigen-binding portion comprises a first heavy chain variable region and a first light chain variable region, and the first antigen-binding portion comprises a first binding domain that binds to the first antigen.

[0308] In a preferred embodiment, the first binding domain comprises a first heavy chain CDR selected from the amino acid sequence of SEQ ID NO. 25, 170, 27 or any variant thereof, and a first light chain CDR selected from the amino acid sequence of SEQ ID NO. 31, 32, 173, 208, 209 or any variant thereof; and

[0309] (b) an antibody or antigen-binding fragment thereof that binds to the CD3 antigen, which is a second antigen-binding portion, wherein the CD3 antigen is the second antigen, the second antigen-binding portion comprises a second heavy chain variable region and a second light chain variable region, the second antigen-binding portion comprises a second binding domain that binds to the second antigen, wherein the second binding domain comprises a second heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO.159 or any variant thereof, a second heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO.160 or any variant thereof, and a second heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO.161 or any variant thereof; and a second light chain CDR1 selected from the amino acid sequence of SEQ ID NO.154 or any variant thereof, a second light chain CDR2 selected from the amino acid sequence of SEQ ID NO.155 or any variant thereof, and a second light chain CDR3 selected from the amino acid sequence of SEQ ID NO.156 or any variant thereof.

[0310] In a preferred embodiment, the second binding domain comprises a second heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 157 or any variant thereof; and a second light chain variable region selected from the amino acid sequence of SEQ ID NO. 152 or any variant thereof.

[0311] In a preferred embodiment, the second heavy chain of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO. 222, and the second light chain of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO. 224.

[0312] In a preferred embodiment, the first binding domain comprises a first heavy chain CDR1 selected from the amino acid sequence SEQ ID NO.25 or any variant thereof, a first heavy chain CDR2 selected from the amino acid sequence SEQ ID NO.170 or any variant thereof, and a first heavy chain CDR3 selected from the amino acid sequence SEQ ID NO.27 or any variant thereof; and a first light chain CDR1 selected from the amino acid sequence SEQ ID NO.173, 208 or any variant thereof, a first light chain CDR2 selected from the amino acid sequence SEQ ID NO.31, 209 or any variant thereof, and a first light chain CDR3 selected from the amino acid sequence SEQ ID NO.32 or any variant thereof.

[0313] In a preferred embodiment, the first light chain CDR of the first binding domain is selected from: the first light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NO.173, 31 and 32, respectively; the first light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NO.208, 209 and 32, respectively; the first light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NO.173, 209 and 32, respectively; the first light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NO.208, 31 and 32, respectively; and the first heavy chain CDR of the first binding domain is selected from: the first heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NO.25, 170 and 27, respectively.

[0314] In a preferred embodiment, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO.168, 202, 232 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO.171, 206, 212, 216, 220, 230 or any variant thereof.

[0315] In a preferred embodiment, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 168 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO. 171 or any variant thereof.

[0316] In a preferred embodiment, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO. 206 or any variant thereof.

[0317] In a preferred embodiment, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO. 212 or any variant thereof.

[0318] In a preferred embodiment, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO. 216 or any variant thereof.

[0319] In a preferred embodiment, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO. 220 or any variant thereof.

[0320] In a preferred embodiment, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 232 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO. 230 or any variant thereof.

[0321] In a preferred embodiment, the first heavy chain of the first antigen-binding moiety is selected from the group consisting of amino acid sequences of SEQ ID NO.196 and 200; and the first light chain of the first antigen-binding moiety is selected from the group consisting of amino acid sequences of SEQ ID NO.198, 204, 210, 214 and 218; more preferably, the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.196; and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.198; more preferably, the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.200; and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.204; more preferably; the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.200 and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.210; more preferably, the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.200; and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.214; more preferably, the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO. NO.200; and the first light chain of the first antigen binding portion comprises the amino acid sequence of SEQ ID NO.218.

[0322] In a more preferred embodiment, the multispecific antibody or antigen-binding fragment thereof further comprises:

[0323] (c) an antibody or antigen-binding fragment thereof that binds to the BCMA antigen, which is a third antigen-binding portion, wherein the BCMA antigen is a third antigen, the third antigen-binding portion comprises a third heavy chain variable region and a third light chain variable region, and the third antigen-binding portion comprises a third binding domain that binds to the third antigen.

[0324] In a preferred embodiment, the third binding domain comprises a third heavy chain variable region selected from the amino acid sequence of SEQ ID NO. 268 or any variant thereof, and a third light chain variable region selected from the amino acid sequence of SEQ ID NO. 267 or any variant thereof.

[0325] In a preferred embodiment, the third antigen binding portion comprises a third heavy chain having an amino acid sequence of SEQ ID NO. 233 or any variant thereof, and a third light chain having an amino acid sequence of SEQ ID NO. 226 or any variant thereof.

[0326] In a preferred embodiment, the multispecific antibody is a trispecific antibody, wherein any two of the first heavy chain variable region, the second heavy chain variable region and the third heavy chain variable region are connected to the constant region, the remaining heavy chain variable region is not connected to the constant region, and the heavy chain variable region not connected to the constant region is connected to any one of the two heavy chain variable regions connected to the constant region, preferably, through a linker; preferably, the heavy chain variable region not connected to the constant region and its corresponding light chain variable region are connected via a linker.

[0327] In a preferred embodiment, the first heavy chain variable region or the first light chain variable region of the first antigen binding moiety is connected to the second heavy chain variable region or the second light chain variable region of the second antigen binding moiety via a linker; preferably, the first heavy chain variable region and the first light chain variable region of the first antigen binding moiety are connected via a linker; more preferably, the first light chain variable region of the first antigen binding moiety is connected to the second heavy chain variable region of the second antigen binding moiety.

[0328] In a preferred embodiment, the first heavy chain variable region or the first light chain variable region of the first antigen binding moiety is connected to the third heavy chain variable region or the third light chain variable region of the third antigen binding moiety via a linker; preferably, the first heavy chain variable region and the first light chain variable region of the first antigen binding moiety are connected via a linker.

[0329] In a preferred embodiment, the linker is a flexible linker.

[0330] In a preferred embodiment, the linker is (GGGGS) n , n=2-5, such as GGGGSGGGGSGGGGS (SEQ ID NO: 231), GSTGSSGKSSEGKG (SEQ ID NO: 273), GGSGGSG (SEQ ID NO: 274), GGGSGGSG (SEQ ID NO: 275);

[0331] In a more preferred embodiment, the trispecific antibody comprises light chain 1, heavy chain 1, light chain 2 and heavy chain 2.

[0332] In a more preferred embodiment, the light chain 1 comprises the third light chain variable region of the amino acid sequence SEQ ID NO.267, the heavy chain 1 comprises the first light chain variable region of the amino acid sequence SEQ ID NO.230, the first heavy chain variable region of the amino acid sequence SEQ ID NO.232 and the third heavy chain variable region of the amino acid sequence SEQ ID NO.268, the light chain 2 comprises the second light chain variable region of the amino acid sequence SEQ ID NO.152, and the heavy chain 2 comprises the second heavy chain variable region of the amino acid sequence SEQ ID NO.157; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.226; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.228; the light chain 2 comprises the amino acid sequence of SEQ ID NO.224; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.222.

[0333] In a more preferred embodiment, the light chain 1 comprises the third light chain variable region of the amino acid sequence SEQ ID NO.267, the heavy chain 1 comprises the first heavy chain variable region of the amino acid sequence SEQ ID NO.232, the first light chain variable region of the amino acid sequence SEQ ID NO.230 and the third heavy chain variable region of the amino acid sequence SEQ ID NO.268, the light chain 2 comprises the second light chain variable region of the amino acid sequence SEQ ID NO.152, and the heavy chain 2 comprises the second heavy chain variable region of the amino acid sequence SEQ ID NO.157; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.226; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.234; the light chain 2 comprises the amino acid sequence of SEQ ID NO.224; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.222.

[0334] In a more preferred embodiment, the light chain 1 comprises a second light chain variable region of the amino acid sequence SEQ ID NO.152, the heavy chain 1 comprises a first light chain variable region of the amino acid sequence SEQ ID NO.230, a first heavy chain variable region of the amino acid sequence SEQ ID NO.232 and a second heavy chain variable region of the amino acid sequence SEQ ID NO.157, the light chain 2 comprises a third light chain variable region of the amino acid sequence SEQ ID NO.267, and the heavy chain 2 comprises a third heavy chain variable region of the amino acid sequence SEQ ID NO.268; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.224; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.236; the light chain 2 comprises the amino acid sequence of SEQ ID NO.226; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.233.

[0335] In a more preferred embodiment, the light chain 1 comprises a second light chain variable region of the amino acid sequence SEQ ID NO.152, the heavy chain 1 comprises a first heavy chain variable region of the amino acid sequence SEQ ID NO.232, a first light chain variable region of the amino acid sequence SEQ ID NO.230 and a second heavy chain variable region of the amino acid sequence SEQ ID NO.157, the light chain 2 comprises a third light chain variable region of the amino acid sequence SEQ ID NO.267, and the heavy chain 2 comprises a third heavy chain variable region of the amino acid sequence SEQ ID NO.268; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.224; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.239; the light chain 2 comprises the amino acid sequence of SEQ ID NO.226; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.233.

[0336] In another aspect, the present disclosure provides a nucleic acid encoding the aforementioned multispecific antibody or antigen-binding fragment thereof.

[0337] In a preferred embodiment, the multispecific antibody is a bispecific antibody, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody is selected from the nucleotide sequences of SEQ ID NO. 169 and SEQ ID NO. 203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion is selected from the nucleotide sequences of SEQ ID NO. 172, SEQ ID NO. 207, SEQ ID NO. 213, SEQ ID NO. 217, and SEQ ID NO. 221; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO. 158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO. 153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody is selected from the nucleotide sequences of SEQ ID NO. 197 and SEQ ID NO. 201, and the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequences of SEQ ID NO. 199, SEQ ID NO. 205, SEQ ID NO. 211, SEQ ID NO. 215, and SEQ ID NO. NO.219; the nucleic acid encoding the second heavy chain of the second antigen binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen binding portion comprises the nucleotide sequence of SEQ ID NO.225.

[0338] In a more preferred embodiment, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.169, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.172; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.197, and the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.199; the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225.

[0339] In a more preferred embodiment, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.207; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, and the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.205; the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225.

[0340] In a more preferred embodiment, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.213; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.211; the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225.

[0341] In a more preferred embodiment, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.217; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.215, the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225.

[0342] In a more preferred embodiment, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.221; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.219, the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225.

[0343] In a preferred embodiment, the multispecific antibody is a trispecific antibody comprising nucleic acids encoding light chain 1, heavy chain 1, light chain 2, and heavy chain 2.

[0344] In a more preferred embodiment, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.269, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.271, the nucleotide sequence of SEQ ID NO.272 and the nucleotide sequence of SEQ ID NO.270, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.153, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.158; more preferably, the nucleic acid encoding the light chain 1 of the trispecific antibody comprises the nucleotide sequence of SEQ ID NO.227, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.229, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.225, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.223.

[0345] In a more preferred embodiment, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.269, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.272, the nucleotide sequence of SEQ ID NO.271 and the nucleotide sequence of SEQ ID NO.270, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.153, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.158; more preferably, the nucleic acid encoding the light chain 1 of the multispecific antibody comprises the nucleotide sequence of SEQ ID NO.227, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.235, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.225, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.223.

[0346] In a more preferred embodiment, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.153, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.271, the nucleotide sequence of SEQ ID NO.272 and the nucleotide sequence of SEQ ID NO.158, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.269, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.270; more preferably, the nucleic acid encoding the light chain 1 of the multispecific antibody comprises the nucleotide sequence of SEQ ID NO.225, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.237, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.227, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.238.

[0347] In a more preferred embodiment, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.153, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.272, the nucleotide sequence of SEQ ID NO.271 and the nucleotide sequence of SEQ ID NO.158, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.269, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.270; more preferably, the nucleic acid encoding the light chain 1 of the multispecific antibody comprises the nucleotide sequence of SEQ ID NO.225, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.240, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.227, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.238.

[0348] In another aspect, the present disclosure provides a vector comprising the aforementioned nucleic acid.

[0349] In another aspect, the present disclosure provides a cell comprising the aforementioned nucleic acid or the aforementioned vector.

[0350] In another aspect, the present disclosure provides a composition comprising the aforementioned GPRC5D antibody or antigen-binding fragment thereof, the aforementioned multispecific antibody or antigen-binding fragment thereof, the aforementioned nucleic acid, the aforementioned vector and / or the aforementioned cell according to claim 1.

[0351] In another aspect, the present disclosure provides a kit comprising the aforementioned GPRC5D antibody or antigen-binding portion thereof, the aforementioned multispecific antibody or antigen-binding portion thereof, the aforementioned nucleic acid, the aforementioned vector, the aforementioned cell and / or the aforementioned composition.

[0352] In another aspect, the present disclosure provides use of the aforementioned GPRC5D antibody or antigen-binding portion thereof, the aforementioned multispecific antibody or antigen-binding portion thereof, the aforementioned nucleic acid, the aforementioned vector, the aforementioned cell and / or the aforementioned composition in the preparation of a medicament or kit for diagnosing, treating or preventing cancer or autoimmune diseases.

[0353] In a preferred embodiment, the cancer is a B-cell related cancer selected from the group consisting of multiple myeloma, malignant plasmacytoma, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Kahler's disease and myeloid leukemia, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ACL), and leukemia. Baxomyeloid leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphocytic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) lymphoma B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma of the elderly, inflammation-related diffuse large B-cell lymphoma B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell-related lymphomas.

[0354] In a preferred embodiment, the autoimmune disease is selected from immune nephropathy, systemic lupus erythematosus or rheumatoid arthritis.

[0355] In one embodiment of the present disclosure, the antibody is administered in a single dose or in multiple doses.

[0356] If the antibody is administered in multiple doses according to the present disclosure, it is preferably administered in at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses or at least 10 doses and preferably up to 30 doses, 25 doses, 20 doses, 15 doses or 10 doses. The doses of the antibody are preferably administered at intervals of at least 7 days, at least 10 days, at least 14 days or at least 20 days. The doses of the antibody are preferably administered at intervals of 7 to 30 days, 10 to 20 days and preferably about 14 days.

[0357] In one aspect, the present disclosure provides the use of a therapeutic agent selected from a monotherapeutic agent or a combination therapeutic agent in the preparation of a medicament for treating a disease associated with cancer in a subject.

[0358] In some embodiments, the monotherapeutic agent is selected from the aforementioned antibodies or antigen-binding portions thereof, nucleic acids, vectors, cells and / or compositions.

[0359] In one aspect, the present disclosure provides a kit comprising the aforementioned antibody or antigen-binding portion thereof, nucleic acid molecule, vector, cell and / or composition.

[0360] The kits disclosed herein include antibodies, fragments, homologues, derivatives thereof, and the like disclosed herein, such as conjugates that are labeled or cytotoxic, and instructions for use of the antibodies, conjugates that kill specific types of cells, and the like. The instructions may include guidance for using the antibodies, conjugates, and the like in vitro, in vivo, or ex vivo. The antibodies may be in liquid form or solid, typically lyophilized. The kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. It is contemplated that the reagents packaged in predetermined amounts are combined with instructions for their use, such as for therapeutic use or for performing diagnostic assays. When the antibody is labeled, such as with an enzyme, the kit may include a substrate and cofactors required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffer or lysis buffer), and the like may also be included. The relative amounts of the various reagents can be varied to provide concentrates of the reagent solutions, which provides user flexibility, saves space, saves reagents, and the like. These reagents may also be provided as a dry powder, usually lyophilized, including excipients which, upon dissolution, provide a solution of the reagent having the appropriate concentration.

[0361] In one aspect, the present disclosure provides use of the antibodies or antigen-binding portions thereof, nucleic acid molecules, vectors, cells and / or compositions in the preparation of medicaments or kits for diagnosing, treating or preventing diseases associated with cancer.

[0362] In addition, the antibodies disclosed herein can also be used in immunoassays, purification methods, and other methods that utilize immunoglobulins or fragments thereof. Such uses are well known in the art.

[0363] Accordingly, the present disclosure also provides a composition comprising the anti-GPRC5D antibody or fragment thereof of the present disclosure, wherein the antibody is conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is a conventional practice in the art.

[0364] The term "pharmaceutical composition" as used in this disclosure refers to a variety of preparations. The formulation containing a therapeutically effective amount of a multivalent antibody is in the form of a sterile liquid solution, liquid suspension, or lyophilized form, optionally containing a stabilizer or excipient.

[0365] It will be understood that the therapeutic agents according to the embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents incorporated into formulations to provide improved transfer, delivery, tolerability, and the like. A wide variety of suitable formulations are available in the pharmacopoeia known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 of Blaug and Seymour therein. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) carriers (e.g., Lipofectin TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be suitable for use in the treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerates the route of administration.

[0366] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, such as a gas such as carbon dioxide, or a nebulizer.

[0367] Administration can also be systemically administered through mucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for the permeability barrier are used in the formulation. Such penetrants are generally known in the art and include detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more of the antibodies can be formulated into ointments, ointments, gels, or creams as are generally known in the art.

[0368] The compounds may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.

[0369] In one embodiment, the antibodies can be prepared with carriers that prevent them from being rapidly eliminated by the body, such as slow-release / controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0370] It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and consistency of dosage. As used herein, dosage unit form refers to physically separable units suitable as unit dosages for the subject to be treated; each unit contains a predetermined amount of one or more of the antibodies described herein calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit form of the embodiments described are dictated by and directly dependent upon the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the limitations inherent in the art of formulating such antibodies for use in treating individuals.

[0371] The pharmaceutical compositions can be placed in a container, pack, or dispenser together with instructions for administration.

[0372] The formulations described herein may also include more than one of the antibodies described, depending on the specific condition to be treated, preferably those with complementary activities that do not negatively affect each other. Alternatively or in addition, the composition may include, for example, an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose. For example, they may be present in combination in a kit or during use.

[0373] In one embodiment, one or more of the antibodies may be administered in combination therapy, i.e., in combination with other agents, such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer and inflammatory diseases). The term "combination" herein refers to administering the agents substantially synchronously, simultaneously, or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at an effective concentration at the treatment site when administration of the second compound begins. In one instance, "combination" may also be the simultaneous inclusion of an antibody of the present disclosure and other therapeutic agents in a kit.

[0374] For example, combination therapies can comprise one or more antibodies described herein co-formulated and / or co-administered with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cytostatic agents, as described in more detail below.) Such combination therapies can advantageously utilize lower dosages of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various monotherapies.

[0375] Experiments in this disclosure have demonstrated that the anti-GPRC5D antibodies provided herein can bind to the human GPRC5D receptor with high affinity, and also bind to cynomolgus monkey GPRC5D with an affinity comparable to that of the human GPRC5D receptor. A novel humanized GPRC5D×CD3 bispecific antibody constructed using the anti-GPRC5D antibodies provided herein has a strong ability to kill low-expressing cells while also being more moderate in activating T cells. In a mouse xenograft tumor model, the GPRC5D×BCMA×CD3 trispecific antibody can simultaneously kill BCMA. + Cells and GPRC5D + The killing effect of bispecific antibodies on myeloma cells is significantly better than that of BCMA×CD3 or GPRC5D×CD3 bispecific antibodies, which helps to overcome the drug resistance caused by a single target and produce more profound and lasting tumor suppression effect in clinical applications.

[0376] For purposes of clarity and conciseness, features are described herein as part of the same or separate embodiments; however, it will be understood that the scope of the present disclosure may include embodiments having a combination of all or some of the described features.

[0377] Example

[0378] Example 1 Generation of anti-GPRC5D antibodies

[0379] (1) Construction of GPRC5D stable cell lines

[0380] Human GPRC5D cDNA (Sino Biological, catalog number: HG24447-UT, the amino acid sequence of which is shown in SEQ ID NO.1) and cynomolgus macaque GPRC5D cDNA (Suzhou Jinweizhi Biosynthesis, the amino acid sequence of which is shown in SEQ ID NO.2) were used as templates to construct lentiviral vector plasmids containing the full-length sequence of human and monkey GPRC5D. According to the instructions of the lentiviral packaging kit (Lenti-Pac HIV Expression Packaging Kit, Gene Copoeia, catalog number: HPK-LvTR-20), the constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells (ATCC CRL-11268) for lentiviral packaging. The culture medium was collected 48 hours after transfection and centrifuged at 500*g for 10 minutes to remove cell debris. The culture supernatant containing lentiviral particles was obtained and filtered through a 0.45μm PES membrane. The supernatant was then dispensed into 1.5mL EP tubes, and 10μL was taken for infection of 1×10 6 HEK293T or CHO cells (ATCC CCL-61) were cultured and 48 hours later, 8 μg / ml puromycin was added to pressure-select the stably transfected cells to obtain monoclonal cell lines HEK293-hGPRC5D (clone 2H3), HEK293-cyGPRC5D (clone 1F11), CHO-hGPRC5D (clone 2D10), and CHO-cyGPRC5D (clone 3E7) expressing human or cynomolgus macaque GPRC5D at high levels. They are hereinafter referred to as HEK293-hGPRC5D, HEK293-cyGPRC5D, CHO-hGPRC5D, and CHO-cyGPRC5D, respectively. The results are shown in Figures 1 and 2.

[0381] The amino acid sequence of human GPRC5D is shown in SEQ ID NO.1.

[0382] The amino acid sequence of cynomolgus monkey GPRC5D is shown in SEQ ID NO. 2.

[0383] (2) Immunization of animals and preparation of hybridomas

[0384] Female Balb / C mice aged 4-6 weeks were selected, and the full-length sequence of human or cynomolgus macaque GPRC5D was used to construct a eukaryotic expression plasmid containing a CMV promoter. The mice were alternately immunized with human and cynomolgus macaque GPRC5D DNA using a gene gun (20 μg plasmid / mouse). After 4-5 immunizations, mice with serum titers that met the requirements were immunized with CHO-hGPRC5D stably transfected cells (5×10 6After 3 days, the mice were killed by cervical dislocation, and the spleen and some peripheral lymph nodes were harvested and ground in DMEM culture medium to obtain a suspension rich in B cells. An appropriate amount of the suspension of lymph nodes and spleen cells was mixed with SP2 / 0 and cell fusion was performed using an electrofusion instrument (BTX). After electrofusion, the cells were fused using ClonaCell TM -HY Medium D (STEMCELL Technologies) for cell plating, IgG + Positive single clones were selected using Clonepix (Molecular Devices) and transferred to 96-well plates. After culturing for 2-3 days, the supernatant was collected to screen hybridoma clones that bound to HEK293-hGPRC5D and HEK293-cyGPRC5D cells.

[0385] Example 2 Screening of hybridoma clones combining human and cynomolgus monkey GPRC5D

[0386] 5 × 10 4 HEK293-hGPRC5D or HEK293-cyGPRC5D cells were centrifuged and the supernatant was discarded. 100 μL / well hybridoma culture supernatant was added and incubated on ice for 60 minutes. After washing away the unbound primary antibody, 50 μL of secondary antibody solution (Anti-mouse IgG, Fc specific-AF647, Jackson Immuno Research) was added to each well and incubated on ice for 30 minutes; the excess secondary antibody was washed away, PBS solution was added to resuspend the cells, and the binding of the antibody to the cells was detected using a flow cytometer (Intellicyte iQue). A total of 290 hybridoma clones that specifically bound to hGPRC5D and cyGPRC5D stably transfected cells were obtained in the initial screening. After enrichment and purification of the above positive clone supernatants, further screening was performed for GPRC5D. + Among the clones that bound to tumor cells, 77 clones showed high binding signals to myeloma cells MM.1S and MM.1R (both purchased from ATCC, catalog numbers CRL-2974 and CRL-2975, respectively).

[0387] RNA was extracted from candidate hybridoma monoclonal cells using the TRNzol lysis method, and then reverse-transcribed to synthesize single-stranded cDNA, which was used as a template to amplify the variable region sequence of the antibody expressed in the hybridoma monoclonal cells. The light chain variable region of the aforementioned antibody was cloned into a vector containing the human light chain constant region, and the heavy chain variable region was cloned into a vector containing the human heavy chain constant region. The plasmids were co-transfected into CHO-S cells, and the intact IgG antibody was expressed and secreted into the culture medium. After 7 days of expression at 37°C, 5% CO₂, and 125 rpm, the supernatant was collected and purified using Protein A chromatography to obtain chimeric IgG. The IgG concentration was determined spectrophotometrically. A control antibody, GC5B596, was synthesized according to US Patent No. 10562968B2, expressed in CHO cells, and purified using Protein A.

[0388] Flow cytometry was used to detect the binding of the above antibodies to human and cynomolgus monkey GPRC5D stably transfected cells. The CHO-hGPRC5D and CHO-cyGPRC5D stably transfected cells prepared in Example 1 were taken and the cell suspension concentration was adjusted to 5×10 5 Cells were plated at 400 μg / ml, 100 μl / well of the cell suspension was added to a 96-well U-shaped plate, centrifuged at 300 g for 5 minutes, the supernatant discarded, and 100 μl of serially diluted antibody (starting concentration 200 nM, 3-fold dilution, 12 steps) was added to each well. The cells were incubated at 4°C for 45 minutes. As the secondary antibody, 50 μl / well of Alexa Fluro 647-labeled goat anti-human IgG Fc (1:300 dilution) was added. The cells were incubated on ice for 45 minutes and detected by flow cytometry.

[0389] Figure 3 shows the binding of anti-GPRC5D chimeric antibodies to GPRC5D stably transfected cell lines. Figure 3A shows the flow cytometric analysis of chimeric antibodies and human GPRC5D stably transfected cells. All chimeras showed significantly better binding activity than GC5B596. Figure 3B shows the flow cytometric analysis of chimeric antibodies and cynomolgus macaque GPRC5D stably transfected cells. Clone 2E1, 2F1, 4B1, 4D3, 8D1, 10B2, 10E8, 15D1, and 19E2 showed strong binding, while GC5B596 only weakly bound to cynomolgus macaque GPRC5D stably transfected cells at the highest concentration of 200 nM. The results are shown in Table 1.

[0390] Table 1 Binding of anti-GPRC5D chimeric antibodies to stably transfected cell lines and tumor cells

[0391] The variable region sequence of the anti-GPRC5D monoclonal antibody is as follows:

[0392] Table 2 Variable region sequences of GPRC5D mouse antibodies

[0393] 1C1

[0394] The VH amino acid sequence is shown in SEQ ID NO.3, its encoding nucleic acid is shown in SEQ ID NO.4, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 5, 6 and 7, respectively.

[0395] Nucleic acid sequence

[0396] The VL amino acid sequence is shown in SEQ ID NO.8, the encoding nucleic acid is shown in SEQ ID NO.9, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.10, 11 and 12, respectively.

[0397] Nucleic acid sequence

[0398] 2E1

[0399] The VH amino acid sequence is shown in SEQ ID NO. 13, its encoding nucleic acid is shown in SEQ ID NO. 14, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 15, 16 and 17, respectively.

[0400] Nucleic acid sequence

[0401] The VL amino acid sequence is shown in SEQ ID NO. 18, the encoding nucleic acid is shown in SEQ ID NO. 19, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 20, 21 and 22, respectively.

[0402] Nucleic acid sequence

[0403] 2F1

[0404] The VH amino acid sequence is shown in SEQ ID NO. 23, its encoding nucleic acid is shown in SEQ ID NO. 24, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 25, 26 and 27, respectively.

[0405] Nucleic acid sequence

[0406] The VL amino acid sequence is shown in SEQ ID NO. 28, the encoding nucleic acid is shown in SEQ ID NO. 29, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 30, 31 and 32, respectively.

[0407] Nucleic acid sequence

[0408] 4B1

[0409] The VH amino acid sequence is shown in SEQ ID NO. 33, its encoding nucleic acid is shown in SEQ ID NO. 34, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 5, 35 and 7, respectively.

[0410] Nucleic acid sequence

[0411] The VL amino acid sequence is shown in SEQ ID NO. 36, its encoding nucleic acid is shown in SEQ ID NO. 37, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 38, 21 and 39, respectively.

[0412] Nucleic acid sequence

[0413] 4D3

[0414] The VH amino acid sequence is shown in SEQ ID NO.40, its encoding nucleic acid is shown in SEQ ID NO.41, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.42, 43 and 44, respectively.

[0415] Nucleic acid sequence

[0416] The VL amino acid sequence is shown in SEQ ID NO.45, its encoding nucleic acid is shown in SEQ ID NO.46, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.47, 48 and 49, respectively.

[0417] Nucleic acid sequence

[0418] 6H1

[0419] The VH amino acid sequence is shown in SEQ ID NO.50, its encoding nucleic acid is shown in SEQ ID NO.51, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.5, 52 and 7, respectively.

[0420] Nucleic acid sequence

[0421] The VL amino acid sequence is shown in SEQ ID NO. 53, its encoding nucleic acid is shown in SEQ ID NO. 54, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 38, 21 and 55, respectively.

[0422] Nucleic acid sequence

[0423] 8C2

[0424] The VH amino acid sequence is shown in SEQ ID NO.56, its encoding nucleic acid is shown in SEQ ID NO.57, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.58, 59 and 60, respectively.

[0425] Nucleic acid sequence

[0426] The VL amino acid sequence is shown in SEQ ID NO.61, its encoding nucleic acid is shown in SEQ ID NO.62, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.63, 64 and 65, respectively.

[0427] Nucleic acid sequence

[0428] 8D1

[0429] The VH amino acid sequence is shown in SEQ ID NO.66, its encoding nucleic acid is shown in SEQ ID NO.67, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.68, 69 and 70, respectively.

[0430] Nucleic acid sequence

[0431] The VL amino acid sequence is shown in SEQ ID NO.71, its encoding nucleic acid is shown in SEQ ID NO.72, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.73, 74 and 75, respectively.

[0432] Nucleic acid sequence

[0433] 10B1

[0434] The VH amino acid sequence is shown in SEQ ID NO.76, its encoding nucleic acid is shown in SEQ ID NO.77, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.241, 78 and 79, respectively.

[0435] Nucleic acid sequence

[0436] The VL amino acid sequence is shown in SEQ ID NO.80, its encoding nucleic acid is shown in SEQ ID NO.81, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.82, 83 and 84, respectively.

[0437] Nucleic acid sequence

[0438] 10B2

[0439] The VH amino acid sequence is shown in SEQ ID NO.40, its encoding nucleic acid is shown in SEQ ID NO.41, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.42, 43 and 44, respectively.

[0440] Nucleic acid sequence

[0441] The VL amino acid sequence is shown in SEQ ID NO.85, its encoding nucleic acid is shown in SEQ ID NO.86, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.47, 48 and 49, respectively.

[0442] Nucleic acid sequence

[0443] 10E8

[0444] The VH amino acid sequence is shown in SEQ ID NO.87, its encoding nucleic acid is shown in SEQ ID NO.88, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.25, 89 and 90, respectively.

[0445] Nucleic acid sequence

[0446] The VL amino acid sequence is shown in SEQ ID NO.91, its encoding nucleic acid is shown in SEQ ID NO.92, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.93, 94 and 95, respectively.

[0447] Nucleic acid sequence

[0448] 12A4

[0449] The VH amino acid sequence is shown in SEQ ID NO.96, its encoding nucleic acid is shown in SEQ ID NO.97, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.98, 99 and 100, respectively.

[0450] Nucleic acid sequence

[0451] The VL amino acid sequence is shown in SEQ ID NO.101, the encoding nucleic acid is shown in SEQ ID NO.102, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.103, 104 and 105, respectively.

[0452] Nucleic acid sequence

[0453] 14D1

[0454] The VH amino acid sequence is shown in SEQ ID NO.106, its encoding nucleic acid is shown in SEQ ID NO.107, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.108, 99 and 109, respectively.

[0455] Nucleic acid sequence

[0456] The VL amino acid sequence is shown in SEQ ID NO.110, the encoding nucleic acid is shown in SEQ ID NO.111, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.112, 104 and 105, respectively.

[0457] Nucleic acid sequence

[0458] 15A6

[0459] The VH amino acid sequence is shown in SEQ ID NO.113, its encoding nucleic acid is shown in SEQ ID NO.114, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.98, 115 and 116, respectively.

[0460] Nucleic acid sequence

[0461] The VL amino acid sequence is shown in SEQ ID NO.110, the encoding nucleic acid is shown in SEQ ID NO.111, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.112, 104 and 105, respectively.

[0462] Nucleic acid sequence

[0463] 15D1

[0464] The VH amino acid sequence is shown in SEQ ID NO.117, its encoding nucleic acid is shown in SEQ ID NO.118, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.119, 99 and 120, respectively.

[0465] Nucleic acid sequence

[0466] The VL amino acid sequence is shown in SEQ ID NO.121, the encoding nucleic acid is shown in SEQ ID NO.122, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.123, 124 and 125, respectively.

[0467] Nucleic acid sequence

[0468] 15H5

[0469] The VH amino acid sequence is shown in SEQ ID NO.126, its encoding nucleic acid is shown in SEQ ID NO.127, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.108, 99 and 116, respectively.

[0470] Nucleic acid sequence

[0471] The VL amino acid sequence is shown in SEQ ID NO. 128, the encoding nucleic acid is shown in SEQ ID NO. 129, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 130, 104 and 105, respectively.

[0472] Nucleic acid sequence

[0473] 17D12

[0474] The VH amino acid sequence is shown in SEQ ID NO.131, its encoding nucleic acid is shown in SEQ ID NO.132, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.108, 133 and 134, respectively.

[0475] Nucleic acid sequence

[0476] The VL amino acid sequence is shown in SEQ ID NO. 135, the encoding nucleic acid is shown in SEQ ID NO. 136, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 112, 104 and 105, respectively.

[0477] Nucleic acid sequence

[0478] 18C3

[0479] The VH amino acid sequence is shown in SEQ ID NO.137, its encoding nucleic acid is shown in SEQ ID NO.138, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.58, 59 and 139, respectively.

[0480] Nucleic acid sequence

[0481] The VL amino acid sequence is shown in SEQ ID NO.61, its encoding nucleic acid is shown in SEQ ID NO.62, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.63, 64 and 65, respectively.

[0482] Nucleic acid sequence

[0483] 18E7

[0484] The VH amino acid sequence is shown in SEQ ID NO.140, its encoding nucleic acid is shown in SEQ ID NO.141, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.98, 99 and 109, respectively.

[0485] Nucleic acid sequence

[0486] The VL amino acid sequence is shown in SEQ ID NO.101, the encoding nucleic acid is shown in SEQ ID NO.102, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.103, 104 and 105, respectively.

[0487] Nucleic acid sequence

[0488] 19E2

[0489] The VH amino acid sequence is shown in SEQ ID NO.140, its encoding nucleic acid is shown in SEQ ID NO.141, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.98, 99 and 109, respectively.

[0490] Nucleic acid sequence

[0491] The VL amino acid sequence is shown in SEQ ID NO. 142, the encoding nucleic acid is shown in SEQ ID NO. 143, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 103, 104 and 144, respectively.

[0492] Nucleic acid sequence

[0493] 22C1

[0494] The VH amino acid sequence is shown in SEQ ID NO.145, its encoding nucleic acid is shown in SEQ ID NO.146, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.25, 147 and 148, respectively.

[0495] Nucleic acid sequence

[0496] The VL amino acid sequence is shown in SEQ ID NO. 149, the encoding nucleic acid is shown in SEQ ID NO. 150, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 93, 151 and 95, respectively.

[0497] Nucleic acid sequence

[0498] Example 3 Construction of GPRC5D×CD3κλ bispecific antibody

[0499] Clones 2E1, 2F1, 4B1, 4D3, 8D1, and 15D1, which showed high affinity for GPRC5D, were selected and their CDRs were transplanted into human germline genes with high homology. These clones were then combined with the humanized anti-CD3e antibody VH9VL5 to construct bispecific antibodies. The Fc portion of the bispecific antibody employed a human IgG4 knob-into-hole structure to achieve heterodimer pairing (Atwell et al., Stable Heterodimers from Remodeling the Domain Interface of a Homodimer using a Phage Display Library, J. Mol. Biol. (1997) 270). Furthermore, mutations at Ser228Pro, Leu235Glu, and Pro329Ala stabilized the hinge region while completely eliminating interactions with Fcγ receptors and C1q.

[0500] The sequence information of the humanized anti-CD3e antibody VH9VL5 is as follows:

[0501] The VL amino acid sequence is shown in SEQ ID NO.152, the encoding nucleic acid is shown in SEQ ID NO.153, and the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.154, 155 and 156, respectively.

[0502] Nucleic acid sequence

[0503] The VH amino acid sequence is shown in SEQ ID NO.157, its encoding nucleic acid is shown in SEQ ID NO.158, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.159, 160 and 161, respectively.

[0504] Nucleic acid sequence

[0505] Table 3 Variable region sequences of GPRC5D humanized antibodies

[0506] Humanized antibody 2E1

[0507] The VH amino acid sequence is shown in SEQ ID NO. 162, its encoding nucleic acid is shown in SEQ ID NO. 163, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 15, 164 and 17, respectively.

[0508] Nucleic acid sequence

[0509] The VL amino acid sequence is shown in SEQ ID NO.165, its encoding nucleic acid is shown in SEQ ID NO.166, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.167, 21 and 22, respectively.

[0510] Nucleic acid sequence

[0511] Humanized antibody 2F1

[0512] The VH amino acid sequence is shown in SEQ ID NO.168, its encoding nucleic acid is shown in SEQ ID NO.169, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.25, 170 and 27, respectively.

[0513] Nucleic acid sequence

[0514] The VL amino acid sequence is shown in SEQ ID NO.171, its encoding nucleic acid is shown in SEQ ID NO.172, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.173, 31 and 32, respectively.

[0515] Nucleic acid sequence

[0516] Humanized antibody 4B1

[0517] The VH amino acid sequence is shown in SEQ ID NO. 174, its encoding nucleic acid is shown in SEQ ID NO. 175, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 5, 176 and 7, respectively.

[0518] Nucleic acid sequence

[0519] The VL amino acid sequence is shown in SEQ ID NO.177, its encoding nucleic acid is shown in SEQ ID NO.178, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.179, 21 and 39, respectively.

[0520] Nucleic acid sequence

[0521] Humanized antibody 4D3

[0522] The VH amino acid sequence is shown in SEQ ID NO.180, its encoding nucleic acid is shown in SEQ ID NO.181, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.42, 182 and 44, respectively.

[0523] Nucleic acid sequence

[0524] The VL amino acid sequence is shown in SEQ ID NO.183, its encoding nucleic acid is shown in SEQ ID NO.184, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.47, 48 and 49, respectively.

[0525] Nucleic acid sequence

[0526] Humanized antibody 8D1

[0527] The VH amino acid sequence is shown in SEQ ID NO. 185, its encoding nucleic acid is shown in SEQ ID NO. 186, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 68, 187 and 70, respectively.

[0528] Nucleic acid sequence

[0529] The VL amino acid sequence is shown in SEQ ID NO.188, its encoding nucleic acid is shown in SEQ ID NO.189, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.190, 74 and 75, respectively.

[0530] Nucleic acid sequence

[0531] Humanized antibody 15D1

[0532] The VH amino acid sequence is shown in SEQ ID NO.191, its encoding nucleic acid is shown in SEQ ID NO.192, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.119, 193 and 120, respectively.

[0533] Nucleic acid sequence

[0534] The VL amino acid sequence is shown in SEQ ID NO.194, its encoding nucleic acid is shown in SEQ ID NO.195, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.1 23, 124 and 125, respectively.

[0535] Nucleic acid sequence

[0536] Plasmids encoding the corresponding antibody fragments were mixed at a ratio of κ light chain:λ light chain:heavy chain 1:heavy chain 2 = 2:2:1:1, mixed with 3 mg / mL PEI, and co-transfected into CHO-S cells. The cells were cultured in 500 mL of CD CHO AGT medium (Gibco #12490-001) at 37°C, 5% CO₂, and 150 rpm. 4% CHO Feed C + supplement (Gibco #A25031-05) was added on days 2, 4, and 6 of transient transfection. When cell viability dropped to approximately 85%, the fermentation broth was harvested and purified by Protein A affinity chromatography. SEC-HPLC indicated a monomer content exceeding 90%. The control GPRC5D×CD3 bispecific antibody GCDB72 was synthesized and assembled according to patent document US10562968B2, and the isotype control anti-KLH×CD3, anti-KLH antibody and anti-CD3e antibody VH9VL5 were constructed according to the WO2022042661A1 method.

[0537] Example 4 Binding activity of GPRC5D×CD3κλ bispecific antibody and TDCC killing

[0538] (1) GPRC5D×CD3κλ bispecific antibody and GPRC5D + Binding of tumor cells

[0539] MM.1R tumor cells in the logarithmic growth phase were taken and 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added and blocked on ice for 30 minutes. The cell suspension concentration was adjusted to 5×10 5Cells were plated at 100 μL per well of a 96-well U-shaped plate and centrifuged at 300 g for 5 minutes. The supernatant was discarded. 100 μL of serially diluted antibody (starting at 200 nM, 3-fold dilution, 10 steps) was added to each well and incubated at 4°C for 60 minutes. The primary antibody was washed away, and 50 μL / well of Alexa Fluor 647-labeled goat anti-human IgG Fc (1:300 dilution) was added and incubated for 20 minutes. After washing once, 50 μL / well of PI was added and incubated for 5 minutes. Detection was performed by flow cytometry. The results are shown in Figure 4. All GPRC5D×CD3κλ bispecific antibodies recognized MM.1R tumor cells. Among them, the binding activity of GPRC5D×CD3κλ bispecific antibodies such as 2E1×CD3, 2F1×CD3, 4B1×CD3, 4D3×CD3 and 8D1×CD3 to tumor cells was significantly stronger than that of GCDB72. The average fluorescence intensity of binding to MM.1R at the maximum concentration of 200 nM was approximately 2.60-4.4 times that of GCDB72 (Table 4).

[0540] (2) TDCC effect mediated by GPRC5D×CD3κλ bispecific antibody

[0541] Freshly isolated PBMCs were mixed with MM.1R tumor cells in logarithmic growth phase at a 10:1 effector:target ratio. 50 μL of serially diluted antibody (7 dilutions, starting at 66.7 nM) was added to each well and incubated at 37°C in 5% CO₂ for 24 hours. After incubation, 50 μL of supernatant was transferred to a new black ELISA plate and 50 μL / well of LDH detection substrate was added. The reaction was terminated after 10 minutes and LDH release was measured. As shown in Figure 5, the 2F1 GPRC5D × CD3κλ bispecific antibody demonstrated potent tumor cell cytotoxicity (EC50 = 0.65 pM), significantly superior to GCDB72 (Table 4).

[0542] Table 4 In vitro activity of GPRC5D×CD3κλ bispecific antibody

[0543] Example 5 Optimization of 2F1 GPRC5D×CD3κλ Humanized Bispecific Antibody

[0544] (1) Construction of 2F1 GPRC5D×CD3κλ bispecific antibody variant

[0545] The 2F1 GPRC5D × CD3κλ bispecific antibody exhibits robust TDCC activity. Different VK1 and VH1 human germline gene families (VK1-33, VK1-39, VH1-46, and VH1-69) were selected to optimize affinity, surface charge distribution, and isoelectric point of the humanized 2F1 antibody, resulting in different 2F1 × CD3κλ bispecific antibody variants (see Tables 5 and 6). Plasmids encoding the GPRC5D and CD3 light and heavy chains were mixed at a ratio of κ light chain:λ light chain:heavy chain 1:heavy chain 2 = 2:2:1:1, mixed with 3 mg / mL PEI, and co-transfected into CHO-S cells. CHO cells were cultured in CD CHO AGT medium at 37°C, 5% CO2, and 150 rpm. 4% CHO Feed C plus supplementation was added on days 2, 4, and 6 after transient transfection. When the cell viability dropped to approximately 85%, the fermentation broth was harvested, filtered, and initially purified by Protein A affinity chromatography. SEC-HPLC showed a monomer content exceeding 90%. Further purification by Capto S ImpAct ion exchange chromatography increased the monomer content to >98-99%.

[0546] Table 5 Light and heavy chain sequences of different 2F1×CD3κλ bispecific antibodies and their variants

[0547] Table 6 Specific sequences of light and heavy chains of different 2F1×CD3κλ bispecific antibodies and their variants

[0548] (2) 2F1 GPRC5D×CD3κλ bispecific antibody and its variants and GPRC5D + Combination of tumor cell lines

[0549] Take MM.1R cells in the logarithmic growth phase and adjust the cell suspension concentration to 5×10 5 100 μL of the solution was added to each well of a 96-well U-shaped plate and centrifuged at 300 g for 5 minutes. The supernatant was discarded, and 100 μL of serially diluted antibody (starting at 1800 nM, 3-fold dilution, 12 steps) was added to each well. The cells were incubated at 4°C for 60 minutes. The primary antibody was washed away, and 50 μL / well of Alexa Fluro 647-conjugated goat anti-human IgG Fc (1:300 dilution) was added. The cells were incubated on ice for 20 minutes. After washing once, 50 μL / well of PI was added and incubated for 5 minutes. The cells were then analyzed by flow cytometry. As shown in Figure 6, the 2F1 GPRC5D × CD3κλ bispecific antibody and its variants all recognized MM.1R cells with slightly reduced affinity compared to the parent 2F1 × CD3 bispecific antibody, but all exhibited higher binding capacity than GCDB72.

[0550] (3) Binding of the 2F1 GPRC5D×CD3κλ bispecific antibody variant to human and cynomolgus macaque GPRC5D stably transfected cell lines

[0551] Take HEK293-hGPRC5D and HEK293-cyGPRC5D stably transfected cells in the logarithmic growth phase and adjust the cell concentration to 5×10 5 Cells were plated at 400 cells / mL, and 100 μL / well of the cell suspension was added to a 96-well U-shaped plate. The plate was centrifuged at 300 g for 5 minutes, and the supernatant was discarded. 100 μL of serially diluted 2F1 GPRC5D×CD3κλ bispecific antibody variants H4K5 and H4K7, as well as GCDB72 (starting at 1800 nM, 3-fold dilution, 12 steps) was added to each well and incubated at 4°C for 60 minutes. As a secondary antibody, 50 μL / well of Alexa Fluro 647-labeled goat anti-human IgG Fc (1:300 dilution) was added and incubated on ice for 20 minutes. After washing once, 50 μL / well of PI solution (1:300) was added and incubated for 5 minutes. Flow cytometry was used for detection. The results are shown in Figure 7 . The 2F1 GPRC5D×CD3κλ bispecific antibody variants bound to human GPRC5D (Figure 7A) and cynomolgus macaque GPRC5D (Figure 7B) with similar affinities (<5-fold) for H4K5 and H4K7, making cynomolgus macaques a relevant species for this study. GCDB72, however, bound weakly to cynomolgus macaque GPRC5D stably transfected cells (Table 6).

[0552] (4) Binding of the 2F1 GPRC5D×CD3κλ bispecific antibody variant to Jurkat T cells

[0553] Take Jurkat cells, a human leukemia T cell line in the logarithmic growth phase, add 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03), and incubate on ice for 30 minutes. Adjust the cell concentration to 5×10 with 4% calf serum. 5Cells were plated at 100 μL per well in a 96-well U-shaped plate, centrifuged at 300 g, and the supernatant removed. 100 μL of serially diluted 2F1 GPRC5D × CD3κλ bispecific antibody variants H4K5 and H4K7, as well as GCDB72 (starting concentration 1800 nM, 3-fold dilution, 10 steps) were added to each well and incubated at 4°C for 60 minutes. Secondary antibodies were added: 50 μL / well of Alexa Fluro 647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI was added for 5 minutes. Flow cytometry was used for detection. The test results are shown in Figure 8. The 2F1 GPRC5D×CD3κλ bispecific antibody variant has comparable binding ability to T cells and is significantly weaker than GCDB72. It helps to reduce the retention of the GPRC5D×CD3κλ bispecific antibody in peripheral lymphoid organs, increase its enrichment at tumor sites, and more gently activate T cells and reduce cytokine release.

[0554] (5) TDCC mediated by 2F1 GPRC5D×CD3κλ bispecific antibody variant

[0555] Freshly isolated PBMCs were mixed with logarithmically growing MM.1R tumor cells at a 10:1 ratio of effector cells to target cells. 50 μL of serially diluted antibodies (7 dilutions, starting at 66.7 nM) were added to each well and incubated at 37°C in 5% CO₂ for 24 hours. After incubation, 50 μL of supernatant was transferred to a new black microplate and 50 μL / well of LDH detection substrate was added. The reaction was terminated after 10 minutes and LDH release was measured. Remaining cells in the wells were washed twice with 4% calf blood and incubated with 100 μg / mL human IgG for 10 minutes. T cell activation detection antibodies (CD25-PE, CD4-APC, CD69-FITC, and CD8-APC) were then added and incubated on ice for 20 minutes. The supernatant was then washed and discarded. 60 μL / well of PI was added and incubated on ice for 5 minutes before analysis by flow cytometry. The test results are shown in Figure 9. The 2F1 GPRC5D×CD3κλ bispecific antibody variant can effectively kill tumor cells by mediating TDCC (Figure 9A). During the killing process, the expression of T cell activation markers CD69 and CD25 was upregulated (Figure 9B), indicating that CD8 + T cells and CD4 + T cells were effectively activated.

[0556] Example 6 Activation of T Cell Activation Pathway by GPRC5D×CD3κλ Bispecific Antibody Variants

[0557] The luciferase reporter plasmid pGL4.30[luc2P / NFAT-RE / Hygro] (Promega, E8481) was transfected into Jurkat cells and 200 μg / ml hygromycin B was added for selection to obtain Jurkat-NFAT luc stably transfected reporter cells. + Target cells, GPRC5D×CD3 antibody and Jurkat-NFAT-luc reporter cells were co-incubated. After the GPRC5D×CD3 antibody bound to the target cells, the other end bound to the CD3 receptor on the surface of Jurkat cells, causing the aggregation of CD3 intracellular signaling motifs. The activation signal was transmitted to the nucleus through the NFAT pathway, ultimately leading to the upregulation of the expression of the reporter gene luciferase.

[0558] Jurkat-NFAT-luc reporter cells and MM.1R cells in the logarithmic growth phase were centrifuged, the supernatant was discarded, and the cells were resuspended to 2×10 6 Target cells were seeded into a 96-well plate at 50 μl / well, centrifuged at 300 g for 5 minutes, and the supernatant discarded. Jurkat-NFAT-luc reporter cells were then seeded into a 96-well plate at 50 μl / well. 50 μl of serially diluted GPRC5D × CD3κλ bispecific antibody variants or control antibody were added to each well (starting at 20 μg / ml, 10-fold dilutions, 10 steps), and incubated at 37°C with 5% CO2 for 6 hours. After incubation, 100 μl of detection reagent was added to each well according to the ONE-Glo Luciferase Assay System instructions. The cells were incubated at room temperature for 3 minutes, and the fluorescence signal was detected (Biotek Synergy HT). Figure 10 shows that activation of Jurkat cells by the GPRC5D × CD3κλ bispecific antibody variants was dependent on the presence of target cells, with an EC50 of approximately 0.66-0.77 nM, which was weaker than the activation of Jurkat T cells by GCDB72 (EC50 = 0.066 nM). Table 7 summarizes the in vitro activity results of the 2F1GPRC5D×CD3κλ bispecific antibody variants.

[0559] Table 7 In vitro activity of 2F1 GPRC5D×CD3κλ bispecific antibody variants

[0560] Example 7 GPRC5D×CD3κλ bispecific antibody variants in immunodeficient mice subcutaneous MM.1S transplanted tumor model

[0561] Female B-NDG mice (Biocytogen Incorporated, Ltd.) aged 6 to 8 weeks were selected and inoculated subcutaneously with 6 × 10 6 MM.1S cells, wait until the tumor grows to 130-140 mm 3The mice were randomly divided into treatment groups of 1.0 mg / kg, 0.1 mg / kg, 0.01 mg / kg and negative control group of KLH×CD3 1 mg / kg. Each mouse was injected intravenously with 1×10 7 PBMC cells were added to the mice. The first dose was administered to mice 6 days later, with a dosing interval of 5 days for a total of 2 doses. The tumor volume and body weight of the mice were monitored. After the experiment, the mice were killed by cervical dislocation, and the tumors were weighed and recorded.

[0562] The results are shown in Figure 11. The in vivo efficacy of the GPRC5D×CD3κλ bispecific antibody variants showed a dose-dependent response, with complete tumor suppression or regression in the high and medium dose groups. At high, medium, and low concentrations, both h2F1H4K5 and h2F1H4K7 exhibited stronger tumor inhibition than GCDB72. Figure 12 demonstrates that tumor-bearing mice tolerated the doses well, with no adverse reactions such as weight loss.

[0563] Example 8 Construction of GPRC5D×BCMA×CD3 triTCE trispecific antibody

[0564] (1) Construction of GPRC5D×BCMA×CD3 triTCE antibody

[0565] The light and heavy chain variable regions of the humanized anti-GPRC5D antibody 2F1H4K5 were linked to the heavy chain N-terminus of the BCMA arm or CD3 arm of the BCMA×CD3κλ005 bispecific antibody (see Chinese patent application CN114573703A) through the GGGGSGGGGSGGGGS flexible polypeptide chain to construct a novel GPRC5D×BCMA×CD3 triTCE trispecific antibody that can simultaneously recognize two tumor antigens and recruit T cells. The GPRC5D×BCMA×CD3 triTCE structure is shown in Figure 13.

[0566] In Tables 8 and 9, the light chain amino acid sequence of BCMA is shown in SEQ ID NO. 226, the encoding nucleic acid thereof is shown in SEQ ID NO. 227, the light chain variable region thereof is shown in SEQ ID NO. 267, and the encoding nucleic acid thereof is shown in SEQ ID NO. 269; the heavy chain amino acid sequence thereof is shown in SEQ ID NO. 233, the encoding nucleic acid thereof is shown in SEQ ID NO. 238, the heavy chain variable region thereof is shown in SEQ ID NO. 268, and the encoding nucleic acid thereof is shown in SEQ ID NO. 270.

[0567] In Tables 8 and 9, the amino acid sequence of the light chain variable region of GPRC5D (GPRC5D-VL) is shown in SEQ ID NO.230, and its encoding nucleic acid is shown in SEQ ID NO 271; the amino acid sequence of the heavy chain variable region of GPRC5D (GPRC5D-VH) is shown in SEQ ID NO.232, and its encoding nucleic acid is shown in SEQ ID NO 272.

[0568] Table 8 GPRC5D×BCMA×CD3 triTCE trispecific antibody sequences

[0569] Table 9 Specific sequences of GPRC5D×BCMA×CD3 triTCE trispecific antibodies

[0570] (2) GPRC5D×BCMA×CD3 triTCE antibody expression and purification

[0571] Plasmids encoding the CD3-GPRC5D scFv arm and the BCMA arm (or plasmids encoding the BCMA-GPRC5D scFv arm and the CD3 arm) were mixed at a ratio of 1:2:1:2 (heavy chain:light chain = 1:2) and transfected into CHO-S cells. After expression at 37°C for 5-6 days, the culture supernatant was collected and purified by Protein A and Capto S ImpAct ion exchange chromatography with a gradient elution using phosphate-citrate / 0.5 M NaCl, pH 7.5. Elution peaks with monomer purity >90% were combined, concentrated, and exchanged into 10 mM acetic acid / 150 mM arginine, pH 4.8 buffer. Mass spectrometry LC-MS (Agilent 1290 Infinity II; Q-TOF 6545XT) confirmed the product as a GPRC5D × BCMA × CD3 triTCE trispecific antibody.

[0572] (3) Binding activity of GPRC5D×BCMA×CD3 triTCE antibody

[0573] (i) BLI affinity determination

[0574] Biofilm interferometry (BLI / Gator Prime) was used to examine the affinity of the GPRC5D×BCMA×CD3 triTCE antibody for recombinant BCMA and CD3 antigens. Biotinylated BCMA or CD3e antigens were captured using an SA probe, and the triTCE antibody was used as the analyte to detect binding and dissociation signals. Results showed that expressing the GPRC5D scFv fused to the N-terminus of the BCMA or CD3 arm had minimal effect on BCMA or CD3 binding (Table 10).

[0575] (ii) Binding to GPRC5D and BCMA stably transfected cell lines

[0576] Using the full-length human BCMA sequence (Sino biological, HG10620-M) as a template, a lentiviral vector plasmid containing the full-length human BCMA sequence was constructed. Following the instructions of the lentiviral packaging kit (Lenti-Pac HIV Expression Packaging Kit, Gene Copoeia, Cat. No.: HPK-LvTR-20), the constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells (ATCC CRL-11268) for lentiviral packaging. 10 μL of the supernatant containing lentiviral particles was used to infect 1×10 6 After 48 hours, 8 μg / ml puromycin was added to HEK293T cells for pressure selection, and HEK-hBCMA cells with high expression of human BCMA were finally isolated. HEK293-hGPRC5D and HEK293-hBCMA cells in the logarithmic growth phase were taken and the cell suspension concentration was adjusted to 5×10 5 Cells were plated at 400 cells / ml, and 100 μL / well of the cell suspension was added to a 96-well U-shaped plate. The cells were centrifuged at 300 g for 5 minutes, and the supernatant was discarded. 100 μL of serially diluted antibodies were added to each well, and the cells were incubated at 4°C for 60 minutes. As a secondary antibody, 50 μL / well of Alexa Fluro 647-conjugated goat anti-human IgG Fc (1:300 dilution) was added, and the cells were incubated on ice for 20 minutes. After washing once, 50 μL / well of PI solution was added, and the cells were incubated for 5 minutes. Flow cytometry was used for analysis. The results showed that the GPRC5D×BCMA×CD3 triTCE trispecific antibody simultaneously bound to both the GPRC5D and BCMA receptors on the cell membrane (Figures 14 and 15), with an affinity (EC50) of approximately 2.7-14.1 nM (Table 10).

[0577] (iii) Binding to Jurkat cells

[0578] Take Jurkat cells in the logarithmic growth phase, add 200 μg / mL mouse IgG, and incubate on ice for 30 minutes. Adjust the cell density to 5×10 with 4% calf serum.5 Cells were plated at 100 μL per well of a 96-well U-shaped plate. The supernatant was removed by centrifugation at 300 g and 100 μL of serially diluted antibodies were added to each well. The cells were incubated at 4°C for 60 minutes. Secondary antibodies were added: Alexa Fluor 647-labeled goat anti-human IgG Fc (1:300 dilution) at 50 μL / well. The cells were incubated on ice for 20 minutes. After washing once, 50 μL / well of PI was added and incubated for 5 minutes. The cells were analyzed by flow cytometry. The results showed that the GPRC5D×BCMA×CD3 triTCE trispecific antibody bound to Jurkat cells with low affinity, and antibody binding did not reach a plateau even at the highest concentration of 200 nM (Figure 16).

[0579] BLI and FACS binding data showed that the GPRC5D×BCMA×CD3 triTCE trispecific antibody bound to BCMA, GPRC5D, and CD3 with essentially comparable affinity compared to the GPRC5D×CD3κλ bispecific antibody or the BCMA×CD3κλ bispecific antibody (Table 10).

[0580] Table 10 Binding activity of GPRC5D×BCMA×CD3 triTCE antibodies

[0581] Example 9 Killing of GPRC5D+ and BCMA+ Cell Lines by GPRC5D×BCMA×CD3 triTCE Trispecific Antibody

[0582] Freshly isolated PBMCs were taken and mixed with target cells NCI-H929 (purchased from ATCC; catalog number: CRL-9068), MM.1S, HEK293-hGPRC5D or HEK293-hBCMA stably transfected cells in the logarithmic growth phase, with effector cells: target cells = 10:1. 50 μL of serially diluted antibodies were added to each well (antibody concentration started from 66.7 nM, 10-fold dilution, 8 gradients), and cultured at 37°C with 5% CO2 for 24 hours. After the incubation period, 50 μL of supernatant was transferred to a new black ELISA plate, 50 μL / well LDH detection substrate was added, the reaction was stopped after 10 minutes, and LDH release was detected. As shown in Figure 17, the GPRC5D×BCMA×CD3 triTCE trispecific antibody can effectively kill BCMA at the same time. + Single positive target cells (Figure 17A), GPRC5D +Single positive target cells (Figure 17B) and myeloma cell lines MM.1S (Figure 17C) and NCI-H929 (Figure 17D) that simultaneously express both GPRC5D and BCMA receptors (Verkleij et al., Preclinical activity and determinants of response of the GPRC5DxCD3 bispecific antibody talquetamab in multiple myeloma, Blood Adv. 2021Apr 27; 5(8); Pillarisetti et al., A T-cell–redirecting bispecific G-protein–coupled receptor class 5member D x CD3 antibody to treat multiple myeloma, Blood. 2020; 135(15); Pillarisetti et al., Teclistamab is an active T cell–redirecting bispecific antibody against B-cell maturation antigen for multiple myeloma, Blood Adv. 2020 Sep22; 4(18)), among which GPRC5D×BCMA×CD3 The tumor cell killing activity mediated by triTCE-v1 and v2 was significantly better than that of the parental GPRC5D×CD3 bispecific antibody or BCMA×CD3 bispecific antibody (Table 11).

[0583] Table 11 TDCC activity of GPRC5D×BCMA×CD3 triTCE

Claims

1. An antibody or antigen-binding fragment thereof that binds to GPRC5D antigen, comprising: (1) a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises: (a) a HCDR1 sequence having the formula: TSYNMH (SEQ ID NO. 5); and (b) a HCDR2 sequence having the formula: YIYPGNGGTNYNQX1FX2X3 (SEQ ID NO. 242), wherein: X1 is K or N, X2 is K or Q, and X3 is A or G; and (c) a HCDR3 sequence having the formula: GYGYRYWYFDV (SEQ ID NO. 7); and (ii) the light chain variable region comprises: (a) a LCDR1 sequence having the formula: X4ASSSVX5X6MH (SEQ ID NO. 243), wherein: X4 is S or R, X5 is S or R, and X6 is Y or F; and (b) a LCDR2 sequence having the formula: DTSKX7AS (SEQ ID NO. 244), wherein: X7 is V or L; and (c) a LCDR3 sequence having the formula: QQWX8X9X 10 PX 11 T (SEQ ID NO. 245), wherein: X8 is S or N, X9 is S or N, X 10 is N, Y or K, X 11 is L or P; or (2) a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises: (a) a HCDR1 sequence having the formula: TGYTMN (SEQ ID NO. 25); and (b) a HCDR2 sequence having the formula: LINPYNGGX 12 X 13 YNX 14 KFX 15 G (SEQ ID NO. 246), wherein: X 12 S or T, X 13 R, S or N, X 14 Q or L, X 15 is a King or Queen; and (c) a HCDR3 sequence having the formula: X 16 X 17 X 18 RX 19 X 20 MDY (SEQ ID NO. 247), wherein: X 16 W or L, X 17 A, P or G, X 18 I or L, X 19 Y or N, X 20 is A or G; and (ii) the light chain variable region comprises: (a) a LCDR1 sequence having the formula: X 21 ASQX 22 X 23 X 24 X 25 NX 26 X 27 (SEQ ID NO. 248), wherein: X 21 is K, R or Q, X 22 N or S, X 23 V or I, X 24 G or S, X 25 T or N, X 26 V or L, X 27 is A or H; and (b) a LCDR2 sequence having the formula: X 28 ASX 29 X 30 X 31 S (SEQ ID NO. 249), wherein: X 28 F, Y or S, X 29 is Y, L or Q, X 30 R, L or S, X 31 is N or 1; and (c) a LCDR3 sequence having the formula: QQX 32 NSX 33 X 34 X 35 T (SEQ ID NO. 250), wherein: X 32 Y or S, X 33 is Y or W, X 34 L or P, X 35 is L or Q; or (3) a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises: (a) a HCDR1 sequence having the formula: TNFPIE (SEQ ID NO. 42); and (b) a HCDR2 sequence having the formula: NFHPYNDVTKYNEKFX 36 G (SEQ ID NO. 251), wherein: X 36 is a King or Queen; and (c) a HCDR3 sequence having the formula: ITRGY (SEQ ID NO. 44); and (ii) the light chain variable region comprises: (a) a LCDR1 sequence having the formula: KSSQSLLYSRNQKNSLA (SEQ ID NO. 47); and (b) a LCDR2 sequence having the formula: WASTRES (SEQ ID NO. 48); and (c) a LCDR3 sequence having the formula: QQYYSYPLT (SEQ ID NO. 49); or (4) a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises: (a) a HCDR1 sequence having the formula: TSYX 37 MY (SEQ ID NO. 252), wherein: X 37 is Y or T; and (b) a HCDR2 sequence having the formula: X 38 INPSX 39 GX 40 TX 41 YNX 42 KFX 43 X 44 (SEQ ID NO.253),Look at: X 38 G or Y, X 39 N or S, X 40 G or Y, X 41 K or N, X 42 Q or E, X 43 K or Q, X 44 is S, D or G; and (c) a HCDR3 sequence having the formula: X 45 X 46 X 47 X 48 X 49 X 50 X 51 YX 52 X 53 X 54 X 55 DY (SEQ ID NO. 254), wherein: X 45 G or W, X 46 G or S, X 47 G or L, X 48 R or N, X 49 E or S, X 50 Y or R, X 51 Y or L, X 52 A or Y, X 53 Y or M, X 54 A or not present, X 55 is M or does not exist; and (ii) the light chain variable region comprises: (a) a LCDR1 sequence having the formula: X 56 ASX 57 X 58 X 59 X 60 X 61 YX 62 X 63 (SEQ ID NO. 255), wherein: X 56 R, Q or S, X 57 Q or S, X 58 D or S, X 59 I or V, X 60 S or not present, X 61 N or S, X 62 For L or M, X 63 is N or Y; and (b) a LCDR2 sequence having the formula: X 64 TSX 65 LX 66 S (SEQ ID NO. 256), wherein: X 64 is Y or G, X 65 R or N, X 66 is H or A; and (c) a LCDR3 sequence having the formula: QQX 67 X 68 SX 69 PX 70 T (SEQ ID NO. 257), wherein: X 67 R or G, X 68 Y or S, X 69 L or Y, X 70 is Y or L; or (5) a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises: (a) a HCDR1 sequence having the formula: TX 71 YVMX 72 (SEQ ID NO. 258), wherein: X 71 S or N, X 72 is Y or H; and (b) a HCDR2 sequence having the formula: YINPYNDGTKYNEX 73 FX 74 G (SEQ ID NO. 259), wherein: X 73 K or R, X 74 is King, Ten or Queen; and (c) a HCDR3 sequence having the formula: GGX 75 X 76 X 77 X 78 X 79 X 80 X 81 X 82 X 83 Y (SEQ ID NO. 260), wherein: X 75 M or V, X 76 L or R, X 77 T or R, X 78 T or Y, X 79 R or F, X 80 S or not present, X 81 L or not present, X 82 F or not present, X 83 is A, T, V, L, or D; and (ii) the light chain variable region comprises: (a) a LCDR1 sequence having the formula: RASQX 84 IX 85 X 86 X 87 X 88 X 89 (SEQ ID NO. 261), wherein: X 84 R, E or D, X 85 G or S, X 86 G or S, X 87 Y or N, X 88 L or F, X 89 is N or S; and (b) a LCDR2 sequence having the formula: AX 90 SX 91 LDS (SEQ ID NO. 262), wherein: X 90 A or T, X 91 is T or S; and (c) a LCDR3 sequence having the formula: LQX 92 X 93 PX 94 T (SEQ ID NO. 263), wherein: X 92 S or N, X 93 Y or F, X 94 is P or Y; or (6) a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises: (a) a HCDR1 sequence having the formula: SSYGMS (SEQ ID NO. 58); and (b) a HCDR2 sequence having the formula: TISSGGSYTYYPDSVKG (SEQ ID NO. 59); and (c) a HCDR3 sequence having the formula: HX 95 X 96 X 97 X 98 X 99 X 100 X 101 X 102 DY (SEQ ID NO. 264), wherein: X 95 G or E, X 96 G or A, X 97 S or R, X 98 S or A, X 99 S or T, X 100 Y or F, X 101 V or not present, X 102 is M or does not exist; and (ii) the light chain variable region comprises: (a) a LCDR1 sequence having the formula: RSSKSLLHSNGNTYLY(SEQ ID NO.63); and (b) a LCDR2 sequence having the formula: RMSNLAS (SEQ ID NO. 64); and (c) a LCDR3 sequence having the formula: MQHLEYPFT (SEQ ID NO. 65); or (7) a heavy chain variable region and a light chain variable region, wherein: (i) the heavy chain variable region comprises: (a) a HCDR1 sequence having the formula: TRYWMH (SEQ ID NO. 15); and (b) a HCDR2 sequence having the formula: EFNPSNGRINYNEKFX 103 X 104 (SEQ ID NO. 265), wherein: X 103 K or Q, X 104 is N or G; and (c) a HCDR3 sequence having the formula: GFAY (SEQ ID NO. 17); and (ii) the light chain variable region comprises: (a) a LCDR1 sequence having the formula: X 105 ASSTVSYIH (SEQ ID NO. 266), wherein: X 105 is S or R; and (b) a LCDR2 sequence having the formula: DTSKLAS (SEQ ID NO. 21); and (c) a LCDR3 sequence having the formula: QQWTTNPWT (SEQ ID NO. 22).

2. The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain CDR1 selected from the group consisting of amino acid sequences of SEQ ID NO. 5, 15, 25, 42, 58, 68, 98, 108, 119, 241, or any variant thereof; a heavy chain CDR2 selected from the group consisting of amino acid sequences of SEQ ID NO. 6, 16, 26, 35, 43, 52, 59, 69, 78, 89, 99, 115, 133, 147, or any variant thereof; a heavy chain CDR3 selected from the group consisting of amino acid sequences of SEQ ID NO. 7, 17, 27, 44, 60, 70, 79, 90, 100, 109, 116, 120, 134, 139, 148, or any variant thereof; and a heavy chain CDR4 selected from the group consisting of amino acid sequences of SEQ ID NO. NO.10, 20, 30, 38, 47, 63, 73, 82, 93, 103, 112, 123, 130, 167, 173, 179, 190, 208 or any variant thereof, a light chain CDR2 selected from the amino acid sequence of SEQ ID NO.11, 21, 31, 48, 64, 74, 83, 94, 104, 124, 151, 209 or any variant thereof, and a light chain CDR3 selected from the amino acid sequence of SEQ ID NO.12, 22, 32, 39, 49, 55, 65, 75, 84, 95, 105, 125, 144 or any variant thereof.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain and a light chain CDR combination selected from the group consisting of: (1) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 6, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 10, 11, and 12, respectively; (2) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 15, 16, and 17, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 20, 21, and 22, respectively; (3) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 26, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 30, 31, and 32, respectively; (4) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 35, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 38, 21, and 39, respectively; (5) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 42, 43, and 44, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 47, 48, and 49, respectively; (6) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 52, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 38, 21, and 55, respectively; (7) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 58, 59, and 60, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 63, 64, and 65, respectively; (8) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 68, 69, and 70, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 73, 74, and 75, respectively; (9) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 241, 78, and 79, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 82, 83, and 84, respectively; (10) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 42, 43, and 44, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 47, 48, and 49, respectively; (11) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 89, and 90, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 93, 94, and 95, respectively; (12) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 99, and 100, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 103, 104, and 105, respectively; (13) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 108, 99, and 109, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 112, 104, and 105, respectively; (14) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 115, and 116, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 112, 104, and 105, respectively; (15) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 119, 99, and 120, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 123, 124, and 125, respectively; (16) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 108, 99, and 116, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 130, 104, and 105, respectively; (17) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 108, 133, and 134, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 112, 104, and 105, respectively; (18) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 58, 59 and 139, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 63, 64 and 65, respectively; (19) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 99, and 109, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 103, 104, and 105, respectively; (20) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 98, 99, and 109, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 103, 104, and 144, respectively; (21) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 147, and 148, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 93, 151, and 95, respectively; (22) heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 15, 164 and 17, respectively, and light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs. 167, 21 and 22, respectively; (23) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 170, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 173, 31, and 32, respectively; (24) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 5, 176, and 7, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 179, 21, and 39, respectively; (25) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 42, 182, and 44, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 47, 48, and 49, respectively; (26) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 68, 187, and 70, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 190, 74, and 75, respectively; (27) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 119, 193, and 120, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 123, 124, and 125, respectively; (28) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 170, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 208, 209, and 32, respectively; (29) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 25, 170, and 27, respectively, and light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs. 173, 209, and 32, respectively; (30) The heavy chain CDR1, CDR2 and CDR3 sequences comprise SEQ ID NOs. 25, 170 and 27, respectively, and the light chain CDR1, CDR2 and CDR3 sequences comprise SEQ ID NOs. 208, 31 and 32, respectively.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy chain variable region selected from the group consisting of the amino acid sequences of SEQ ID NO. 3, 13, 23, 33, 40, 50, 56, 66, 76, 87, 96, 106, 113, 117, 126, 131, 140, 145, 162, 168, 174, 180, 185, 191, 202, 232, or any variant thereof, and a light chain variable region selected from the group consisting of the amino acid sequences of SEQ ID NO. 8, 18, 28, 36, 45, 53, 61, 71, 80, 85, 91, 101, 110, 121, 128, 135, 142, 149, 165, 171, 177, 183, 188, 194, 206, 212, 216, 220, 230, or any variant thereof; Preferably, it comprises a heavy chain variable region having an amino acid sequence of SEQ ID NO.3 or any variant thereof, and a light chain variable region having an amino acid sequence of SEQ ID NO.8 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.13 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.18 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.23 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.28 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO. 33 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO. 36 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.40 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.45 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.50 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.53 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.56 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.61 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.66 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.71 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.76 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.80 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.40 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.85 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.87 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.91 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.96 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.101 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.106 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.110 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.113 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.110 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.117 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.121 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.126 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.128 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.131 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.135 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.137 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.61 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.140 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.101 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.140 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.142 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.145 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.149 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.162 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.165 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.168 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.171 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.174 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.177 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.180 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.183 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.185 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.188 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.191 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.194 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.202 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.206 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.202 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.212 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.202 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.216 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.202 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.220 or any variant thereof; Preferably, it comprises a heavy chain variable region of the amino acid sequence SEQ ID NO.232 or any variant thereof, and a light chain variable region of the amino acid sequence SEQ ID NO.230 or any variant thereof; Preferably, the antibody or antigen-binding fragment thereof is humanized.

5. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; Preferably, the nucleic acid comprises an antibody heavy chain variable region nucleic acid sequence selected from SEQ ID NO. 4, 14, 24, 34, 41, 51, 57, 67, 77, 88, 97, 107, 114, 118, 127, 132, 138, 141, 146, 163, 169, 175, 181, 186, 192, 203, or any variant thereof, and an antibody light chain variable region nucleic acid sequence selected from SEQ ID NO. 9, 19, 29, 37, 46, 54, 62, 72, 81, 86, 92, 102, 111, 122, 129, 136, 143, 150, 166, 172, 178, 184, 189, 195, 207, 213, 217, 221, or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 4 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 9 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 14 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 19 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 24 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 29 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 34 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 37 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.41 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.46 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.51 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.54 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.57 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.62 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.67 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.72 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 77 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 81 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.41 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.86 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.88 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.92 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.97 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.102 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.107 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.111 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.114 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.111 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.118 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.122 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 127 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 129 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 132 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 136 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.138 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.62 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.141 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.102 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.141 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.143 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO.146 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO.150 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 163 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 166 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 169 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 172 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 175 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 178 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 181 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 184 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 186 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 189 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 192 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 195 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 207 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 213 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 217 or any variant thereof; More preferably, the nucleic acid comprises a heavy chain variable region nucleotide sequence of SEQ ID NO. 203 or any variant thereof, and a light chain variable region nucleotide sequence of SEQ ID NO. 221 or any variant thereof.

6. A multispecific antibody or antigen-binding fragment thereof, comprising at least: (a) an antibody or antigen-binding fragment thereof that binds to the GPRC5D antigen according to any one of claims 1 to 4, which is a first antigen-binding portion, wherein the GPRC5D antigen is a first antigen, and the first antigen-binding portion comprises a first heavy chain variable region and a first light chain variable region, and the first antigen-binding portion comprises a first binding domain that binds to the first antigen; Preferably, the first binding domain comprises a first heavy chain CDR selected from the amino acid sequence of SEQ ID NO. 25, 170, 27 or any variant thereof, and a first light chain CDR selected from the amino acid sequence of SEQ ID NO. 31, 32, 173, 208, 209 or any variant thereof; and (b) an antibody or antigen-binding fragment thereof that binds to the CD3 antigen, which is a second antigen-binding portion, wherein the CD3 antigen is a second antigen, the second antigen-binding portion comprises a second heavy chain variable region and a second light chain variable region, and the second antigen-binding portion comprises a second binding domain that binds to the second antigen, wherein: The second binding domain comprises a second heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO.159 or any variant thereof, a second heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO.160 or any variant thereof, and a second heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO.161 or any variant thereof; and a second light chain CDR1 selected from the amino acid sequence of SEQ ID NO.154 or any variant thereof, a second light chain CDR2 selected from the amino acid sequence of SEQ ID NO.155 or any variant thereof, and a second light chain CDR3 selected from the amino acid sequence of SEQ ID NO.156 or any variant thereof; Preferably, the second binding domain comprises a second heavy chain variable region selected from the amino acid sequence SEQ ID NO.157 or any variant thereof; and a second light chain variable region selected from the amino acid sequence SEQ ID NO.152 or any variant thereof; Preferably, the second heavy chain of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO.222, and the second light chain of the second antigen binding moiety comprises the amino acid sequence of SEQ ID NO.224; Preferably, the first binding domain comprises a first heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO.25 or any variant thereof, a first heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO.170 or any variant thereof, and a first heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO.27 or any variant thereof; and a first light chain CDR1 selected from the amino acid sequence of SEQ ID NO.173, 208 or any variant thereof, a first light chain CDR2 selected from the amino acid sequence of SEQ ID NO.31, 209 or any variant thereof, and a first light chain CDR3 selected from the amino acid sequence of SEQ ID NO.32 or any variant thereof; Preferably, the first light chain CDR of the first binding domain is selected from the group consisting of: first light chain CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs. 173, 31, and 32, respectively; first light chain CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs. 208, 209, and 32, respectively; first light chain CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs. 173, 209, and 32, respectively; first light chain CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs. 208, 31, and 32, respectively; and the first heavy chain CDR of the first binding domain is selected from the group consisting of: first heavy chain CDR1, CDR2, and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs. 25, 170, and 27, respectively; Preferably, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO.168, 202, 232 or any variant thereof; and a first light chain variable region selected from the amino acid sequence of SEQ ID NO.171, 206, 212, 216, 220, 230 or any variant thereof; Preferably, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence SEQ ID NO.168 or any variant thereof; and a first light chain variable region selected from the amino acid sequence SEQ ID NO.171 or any variant thereof; Preferably, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence SEQ ID NO.202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence SEQ ID NO.206 or any variant thereof; Preferably, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence SEQ ID NO.202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence SEQ ID NO.212 or any variant thereof; Preferably, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence SEQ ID NO.202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence SEQ ID NO.216 or any variant thereof; Preferably, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence SEQ ID NO.202 or any variant thereof; and a first light chain variable region selected from the amino acid sequence SEQ ID NO.220 or any variant thereof; Preferably, the first binding domain comprises a first heavy chain variable region selected from the amino acid sequence SEQ ID NO.232 or any variant thereof; and a first light chain variable region selected from the amino acid sequence SEQ ID NO.230 or any variant thereof; Preferably, the first heavy chain of the first antigen-binding moiety is selected from the group consisting of amino acid sequences of SEQ ID NO.196 and 200; and the first light chain of the first antigen-binding moiety is selected from the group consisting of amino acid sequences of SEQ ID NO.198, 204, 210, 214 and 218; more preferably, the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.196; and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.198; more preferably, the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.200; and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.204; more preferably,; the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.200 and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.210; more preferably, the first heavy chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.200; and the first light chain of the first antigen-binding moiety comprises the amino acid sequence of SEQ ID NO.214; more preferably, The first heavy chain of the first antigen binding moiety comprises the amino acid sequence of SEQ ID NO.200; and the first light chain of the first antigen binding moiety comprises the amino acid sequence of SEQ ID NO.218; More preferably, the multispecific antibody or antigen-binding fragment thereof further comprises: (c) an antibody or antigen-binding fragment thereof that binds to a BCMA antigen, which is a third antigen-binding portion, wherein the BCMA antigen is a third antigen, the third antigen-binding portion comprises a third heavy chain variable region and a third light chain variable region, and the third antigen-binding portion comprises a third binding domain that binds to the third antigen; Preferably, the third binding domain comprises a third heavy chain variable region selected from the amino acid sequence of SEQ ID NO.268 or any variant thereof, and a third light chain variable region selected from the amino acid sequence of SEQ ID NO.267 or any variant thereof; Preferably, the third antigen-binding portion comprises a third heavy chain having an amino acid sequence of SEQ ID NO.233 or any variant thereof, and a third light chain having an amino acid sequence of SEQ ID NO.226 or any variant thereof; Preferably, the multispecific antibody is a trispecific antibody, wherein any two of the first heavy chain variable region, the second heavy chain variable region, and the third heavy chain variable region are connected to a constant region, and the remaining heavy chain variable region is not connected to a constant region, and the heavy chain variable region not connected to the constant region is connected to any one of the two heavy chain variable regions connected to the constant region, preferably, via a linker; preferably, the heavy chain variable region not connected to the constant region is connected to its corresponding light chain variable region via a linker; Preferably, the first heavy chain variable region or the first light chain variable region of the first antigen binding moiety is connected to the second heavy chain variable region or the second light chain variable region of the second antigen binding moiety via a linker; preferably, the first heavy chain variable region and the first light chain variable region of the first antigen binding moiety are connected via a linker; more preferably, the first light chain variable region of the first antigen binding moiety is connected to the second heavy chain variable region of the second antigen binding moiety; Preferably, the first heavy chain variable region or the first light chain variable region of the first antigen binding moiety is connected to the third heavy chain variable region or the third light chain variable region of the third antigen binding moiety via a linker; preferably, the first heavy chain variable region and the first light chain variable region of the first antigen binding moiety are connected via a linker; Preferably, the linker is a flexible linker; more preferably, the linker is (GGGGS) n , n=2-5, such as GGGGSGGGGSGGGGS (SEQ ID NO: 231), GSTGSSGKSSEGKG (SEQ ID NO: 273), GGSGGSG (SEQ ID NO: 274), GGGSGGSG (SEQ ID NO: 275); More preferably, the trispecific antibody comprises light chain 1, heavy chain 1, light chain 2, and heavy chain 2; More preferably, the light chain 1 comprises the third light chain variable region of the amino acid sequence SEQ ID NO.267, the heavy chain 1 comprises the first light chain variable region of the amino acid sequence SEQ ID NO.230, the first heavy chain variable region of the amino acid sequence SEQ ID NO.232, and the third heavy chain variable region of the amino acid sequence SEQ ID NO.268, the light chain 2 comprises the second light chain variable region of the amino acid sequence SEQ ID NO.152, and the heavy chain 2 comprises the second heavy chain variable region of the amino acid sequence SEQ ID NO.157; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.226; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.228; the light chain 2 comprises the amino acid sequence of SEQ ID NO.224; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.222; More preferably, the light chain 1 comprises the third light chain variable region of the amino acid sequence SEQ ID NO.267, the heavy chain 1 comprises the first heavy chain variable region of the amino acid sequence SEQ ID NO.232, the first light chain variable region of the amino acid sequence SEQ ID NO.230, and the third heavy chain variable region of the amino acid sequence SEQ ID NO.268, the light chain 2 comprises the second light chain variable region of the amino acid sequence SEQ ID NO.152, and the heavy chain 2 comprises the second heavy chain variable region of the amino acid sequence SEQ ID NO.157; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.226; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.234; the light chain 2 comprises the amino acid sequence of SEQ ID NO.224; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.222; More preferably, the light chain 1 comprises the second light chain variable region of the amino acid sequence SEQ ID NO.152, the heavy chain 1 comprises the first light chain variable region of the amino acid sequence SEQ ID NO.230, the first heavy chain variable region of the amino acid sequence SEQ ID NO.232, and the second heavy chain variable region of the amino acid sequence SEQ ID NO.157, the light chain 2 comprises the third light chain variable region of the amino acid sequence SEQ ID NO.267, and the heavy chain 2 comprises the third heavy chain variable region of the amino acid sequence SEQ ID NO.268; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.224; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.236; the light chain 2 comprises the amino acid sequence of SEQ ID NO.226; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.233; More preferably, the light chain 1 comprises a second light chain variable region of the amino acid sequence SEQ ID NO.152, the heavy chain 1 comprises a first heavy chain variable region of the amino acid sequence SEQ ID NO.232, a first light chain variable region of the amino acid sequence SEQ ID NO.230 and a second heavy chain variable region of the amino acid sequence SEQ ID NO.157, the light chain 2 comprises a third light chain variable region of the amino acid sequence SEQ ID NO.267, and the heavy chain 2 comprises a third heavy chain variable region of the amino acid sequence SEQ ID NO.268; more preferably, the light chain 1 comprises the amino acid sequence of SEQ ID NO.224; the heavy chain 1 comprises the amino acid sequence of SEQ ID NO.239; the light chain 2 comprises the amino acid sequence of SEQ ID NO.226; and the heavy chain 2 comprises the amino acid sequence of SEQ ID NO.

233.

7. A nucleic acid encoding the multispecific antibody or antigen-binding fragment thereof according to claim 6; Preferably, the multispecific antibody is a bispecific antibody, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody is selected from the nucleotide sequences of SEQ ID NO.169 and SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion is selected from the nucleotide sequences of SEQ ID NO.172, SEQ ID NO.207, SEQ ID NO.213, SEQ ID NO.217 and SEQ ID NO.221; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody is selected from the nucleotide sequences of SEQ ID NO.197 and SEQ ID NO.201, and the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequences of SEQ ID NO.199, SEQ ID NO.205, SEQ ID NO.211, SEQ ID NO.215 and SEQ ID NO. NO.219; the nucleic acid encoding the second heavy chain of the second antigen-binding moiety comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding moiety comprises the nucleotide sequence of SEQ ID NO.225; More preferably, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.169, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.172; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.197, and the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.199; the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225; More preferably, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.207; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, and the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.205; the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225; More preferably, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.213; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.211; the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225; More preferably, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.217; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.215, the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225; More preferably, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.203, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.221; the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.158, and the nucleic acid encoding the second light chain variable region of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.153; more preferably, the nucleic acid encoding the first heavy chain of the first antigen-binding portion of the bispecific antibody comprises the nucleotide sequence of SEQ ID NO.201, the nucleic acid encoding the first light chain of the first antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.219, the nucleic acid encoding the second heavy chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.223, and the nucleic acid encoding the second light chain of the second antigen-binding portion comprises the nucleotide sequence of SEQ ID NO.225; Preferably, the multispecific antibody is a trispecific antibody comprising nucleic acids encoding light chain 1, heavy chain 1, light chain 2, and heavy chain 2; More preferably, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.269, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.271, the nucleotide sequence of SEQ ID NO.272 and the nucleotide sequence of SEQ ID NO.270, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.153, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.158; more preferably, the nucleic acid encoding the light chain 1 of the trispecific antibody comprises the nucleotide sequence of SEQ ID NO.227, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.229, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.225, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.223; More preferably, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.269, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.272, the nucleotide sequence of SEQ ID NO.271 and the nucleotide sequence of SEQ ID NO.270, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.153, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.158; more preferably, the nucleic acid encoding the light chain 1 of the multispecific antibody comprises the nucleotide sequence of SEQ ID NO.227, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.235, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.225, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.223; More preferably, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.153, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.271, the nucleotide sequence of SEQ ID NO.272, and the nucleotide sequence of SEQ ID NO.158, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.269, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.270; more preferably, the nucleic acid encoding the light chain 1 of the multispecific antibody comprises the nucleotide sequence of SEQ ID NO.225, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.237, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.227, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.238; More preferably, the nucleic acid encoding the light chain 1 comprises the nucleotide sequence of SEQ ID NO.153, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.272, the nucleotide sequence of SEQ ID NO.271 and the nucleotide sequence of SEQ ID NO.158, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.269, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.270; more preferably, the nucleic acid encoding the light chain 1 of the multispecific antibody comprises the nucleotide sequence of SEQ ID NO.225, the nucleic acid encoding the heavy chain 1 comprises the nucleotide sequence of SEQ ID NO.240, the nucleic acid encoding the light chain 2 comprises the nucleotide sequence of SEQ ID NO.227, and the nucleic acid encoding the heavy chain 2 comprises the nucleotide sequence of SEQ ID NO.

238.

8. A vector comprising the nucleic acid according to claim 5 or 7.

9. A cell comprising the nucleic acid of claim 5 or 7 or the vector of claim 8.

10. A composition comprising the GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the multispecific antibody or antigen-binding fragment thereof according to claim 6, the nucleic acid according to claim 5 or 7, the vector according to claim 8 and / or the cell according to claim 9.

11. A kit comprising the GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the multispecific antibody or antigen-binding fragment thereof according to claim 6, the nucleic acid according to claim 5 or 7, the vector according to claim 8, the cell according to claim 9, and / or the composition according to claim 10.

12. Use of the GPRC5D antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, the multispecific antibody or antigen-binding fragment thereof according to claim 6, the nucleic acid according to claim 5 or 7, the vector according to claim 8, the cell according to claim 9, and / or the composition according to claim 10 in the preparation of a medicament or kit for diagnosing, treating, or preventing cancer or autoimmune diseases; Preferably, the cancer is a B-cell related cancer selected from the group consisting of multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myeloid leukemia, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphocytic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma lymphoma, lymphoplasmacytic lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma of the elderly, inflammation-associated diffuse large B-cell lymphoma B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell-related lymphomas; Preferably, the autoimmune disease is selected from immune nephropathy, systemic lupus erythematosus or rheumatoid arthritis.

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