Anti-GPRC5DxBCMAxCD3 trispecific antibodies and uses thereof

Abstract

The present invention relates to a trispecific antigen-binding protein, more specifically, a trispecific antibody that specifically binds to two tumor antigens, GPRC5D and BCMA, and the T-cell surface antigen CD3, as well as pharmaceutical compositions comprising the same, methods for preparing and using the same.

Description

[Technical field]

[0001] The present invention relates to a trispecific antigen-binding protein, more specifically, a trispecific antibody that specifically binds to two tumor antigens, GPRC5D and BCMA, and the T-cell surface antigen CD3, as well as pharmaceutical compositions comprising the same, methods for preparing and using the same. [Background technology]

[0002] T cell bispecific antibodies (BsAb) have been used in the treatment of tumors. Such antibodies can form synapses between cytotoxic T lymphocytes and tumor cells, inducing the destruction of tumor cells. BsAb usually involve dual targeting against tumor-associated antigens (TAA) and T cell surface antigens (also called T cell engaging antigens, TEA). However, BsAb-based therapeutic regimens typically depend on the distribution of tumor-associated antigens in the tumor cells to be treated. This has led to selectivity of treatment for patient populations and limited therapeutic potential. Moreover, studies have shown that therapeutic formats targeting a single TAA site may limit the efficacy of treatment, as it may cause disease recurrence due to tumor escape mechanisms.

[0003] Trispecific and / or tetraspecific antibodies have been proposed to be developed to efficiently recruit immune cells to tumor sites and improve therapeutic efficacy. For this reason, various multivalent, multispecific antibody formats have been reported in recent years. However, the diverse requirements of various therapeutic products in terms of therapeutic functionality and therapeutic behavior dictate that there is no "optimal format" that can be applied to most different desired molecular combinations. Therefore, when achieving multispecificity, various factors must be considered, such as the spatial distribution or size of different target antigens and the expression density of tumor-associated antigens on the tumor cell surface. In many cases, an effective antibody format that combines a particular target antigen needs to be identified by producing and comparing the functionality of different antibody formats. Summary of the Invention

[0004] Because there is no correlation between the expression of GPRC5D and BCMA in tumors, the inventors have proposed that by combining multispecific antibodies targeting GPRC5D and BCMA, it is possible to cover both GPRC5D-positive tumor patients and BCMA-positive tumor patients, while avoiding relapse due to loss of a single antigen, thereby achieving the goals of increasing patient coverage and improving therapeutic efficacy.

[0005] To achieve this goal, the present inventors have proposed a trispecific antibody targeting GPRC5D / BCMA / CD3 and have studied multiple GPRC5DxBCMAxCD3 formats in detail. By adjusting the combination method and distance between two TAA antigens (GPRC5D and BCMA) and T cell surface antigen (CD3), and by adjusting the action of T cell engager antibodies in mediating T cell activation and killing effects, the present inventors successfully achieve multi-target combination and reduce the need for multiple drug combinations. On this basis, the Knob-in-Hole technology is further used by the Fc segment to reduce the mismatch of heavy chains, while the problem of light chain mismatch is solved by means of extracellular redox. The antibody of the present invention also has good preparation potential and good drug discovery potential.

[0006] Thus, in one aspect, the present invention provides a trispecific Y-shaped antibody molecule having the above-mentioned advantages, comprising antigen-binding sites that specifically bind to GPRC5D, BCMA and CD3, and comprising two antibody arms, a stem located at the C-terminus of the antibody arms, and a conjugated moiety at the C-terminus of the antibody arms and / or stem.

[0007] In a further aspect, the present invention provides an antibody molecule that specifically binds to GPRC5D, comprising a combination of CDR sequences selected from SEQ ID NOs: 1 to 6, 7 to 12, 13 to 18, or 19 to 24 in the order of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3. The antibody molecule of the present invention binds to GPRC5D with high affinity and blocks GPRC5D-mediated signaling in cells.

[0008] In further aspects, the present invention also provides nucleic acids, vectors and host cells encoding the antibody molecules of the invention, as well as uses of the antibody molecules of the invention, in particular methods and uses for treating GPRC5D-positive and / or BCMA-positive tumours, such as multiple myeloma. [Brief description of the drawings]

[0009] Preferred embodiments of the invention, which are described in detail below, are better understood when read in conjunction with the following drawings: For the purposes of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, but it should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Figure 1A] The structures of trispecific antibody molecules designed using Format_1, 2, 6 and 7 are shown diagrammatically. [Figure 1B] The structures of trispecific antibody molecules designed using Format_1, 2, 6 and 7 are shown diagrammatically. [Figure 1C] The structures of trispecific antibody molecules designed using Format_1, 2, 6 and 7 are shown diagrammatically. [Figure 1D] The structures of trispecific antibody molecules designed using Format_1, 2, 6 and 7 are shown diagrammatically. [Figure 2A] Schematic representation of the structure of a control bispecific antibody molecule used in the examples. [Figure 2B] Schematic representation of the structure of a control bispecific antibody molecule used in the examples. [Figure 2C] Schematic representation of the structure of a control bispecific antibody molecule used in the examples. [Figure 3A] 1 shows detection of exemplary antibodies TS-F2-1 and -2 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 3B] 1 shows detection of exemplary antibodies TS-F2-1 and -2 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 4A] 1 shows detection of exemplary antibodies TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 4B] 1 shows detection of exemplary antibodies TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 4C] 1 shows detection of exemplary antibodies TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 4D] 1 shows detection of exemplary antibodies TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 5A] Detection of Exemplary Antibodies TS-F2-4 and 5-Mediated T Cell Activation (AD) by the Jurkat-NFAT-Luc Reporting System; [Figure 5B] Detection of Exemplary Antibodies TS-F2-4 and 5-Mediated T Cell Activation (AD) by the Jurkat-NFAT-Luc Reporting System; [Figure 5C] Detection of Exemplary Antibodies TS-F2-4 and 5-Mediated T Cell Activation (AD) by the Jurkat-NFAT-Luc Reporting System; [Figure 5D] Detection of Exemplary Antibodies TS-F2-4 and 5-Mediated T Cell Activation (AD) by the Jurkat-NFAT-Luc Reporting System; [Figure 5E] 1 shows detection of exemplary antibodies TS-F2-4, 5 and 6 mediated T cell activation (EF) by the Jurkat-NFAT-Luc reporting system. [Figure 5F]1 shows detection of exemplary antibodies TS-F2-4, 5 and 6 mediated T cell activation (EF) by the Jurkat-NFAT-Luc reporting system. [Figure 6A] 1 shows detection of exemplary antibodies TS-F7-1 and 7-2 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 6B] 1 shows detection of exemplary antibodies TS-F7-1 and 7-2 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 6C] 1 shows detection of exemplary antibodies TS-F7-1 and 7-2 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 6D] 1 shows detection of exemplary antibodies TS-F7-1 and 7-2 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 7] 1 shows detection of exemplary antibodies TS-F7-3 and 7-4 mediated T cell activation by the Jurkat-NFAT-Luc reporting system. [Figure 8] 1 shows an exemplary antibody-mediated activation of CD4+ T cells in PBMCs. [Figure 9] An exemplary antibody-mediated activation of CD8+ T cells in PBMCs (B) is shown. [Figure 10] 1 shows an exemplary antibody-mediated PBMC killing effect on H929 cells. [Figure 11A] 1 shows an exemplary antibody-induced PBMC cytokine release associated with the NCl-H929 cell killing process. [Figure 11B] 1 shows an exemplary antibody-induced PBMC cytokine release associated with the NCl-H929 cell killing process. [Figure 11C] 1 shows an exemplary antibody-induced PBMC cytokine release associated with the NCl-H929 cell killing process. [Figure 11D] 1 shows an exemplary antibody-induced PBMC cytokine release associated with the NCl-H929 cell killing process. [Figure 12]1 shows the tumor suppressive effect of exemplary antibodies in an H929 tumor-bearing humanized mouse model. [Figure 13] 1 shows the expression levels of GPRC5D and BCMA on the surface of different multiple myeloma cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Unless otherwise specified, all technical and scientific terms used herein have the usual meanings understood by those skilled in the art of the present invention. All publications, patent applications, patents or other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods and examples described herein are illustrative and not intended to be limiting. Other features, objects and advantages of the present invention will become apparent from the specification and drawings, and the appended claims.

[0011] I. Definition The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​from 5% less than the number or numerical value specified as the lower limit to 5% more than the number or numerical value specified as the upper limit.

[0012] As used herein, the terms "comprise" or "comprise" mean the inclusion of a stated element, integer or step, but not the exclusion of any other elements, integers or steps.

[0013] The term "antibody" is used herein in the broadest sense to mean a protein that contains an antigen-binding site and includes natural and artificial antibodies of various structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single chain antibodies, complete antibodies and antibody fragments.

[0014] The terms "whole antibody", "full length antibody", "complete antibody" and "intact antibody" refer to a naturally occurring glycoprotein comprising at least two heavy chains (H) and two light chains (L) connected to each other via disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further divided into hypervariable regions (complementarity determining regions (CDRs)) with conserved regions (framework regions (FRs)) between them. Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in binding between an antibody and an antigen, but exhibits various effector functions.

[0015] The term "antigen-binding fragment" refers to a portion or fragment of an intact or complete antibody that is less than the number of amino acid residues of the intact or complete antibody and can bind to an antigen or can bind to an antigen competitively with the intact antibody (i.e., the intact antibody from which the antigen-binding fragment is derived). Antigen-binding fragments can be prepared by recombinant DNA technology or by cleaving an intact antibody by enzymatic or chemical means. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, and single domain antibody (sdAb). The Fab fragment is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, for example, a Fab fragment is obtained by digesting a complete antibody with papain. Also, F(ab')2 produced by digesting a complete antibody below the disulfide bond in the hinge region with pepsin is a dimer of Fab' and is a bivalent antibody fragment. F(ab')2 can be reduced by breaking the disulfide bonds in the hinge region under neutral conditions, which converts the F(ab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (for a detailed description of other antibody fragments, see Fundamental Immunology, edited by WE Paul, Raven Press, NY (1993)). The Fv fragment consists of the single-arm VL and VH domains of an antibody. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be connected by a synthetic connecting peptide that can produce the two domains as a single protein chain by recombinant methods, in which the VL and VH regions pair to form a single chain Fv. The antibody fragment can be obtained by chemical methods, DNA recombinant methods, or protein enzyme digestion methods.

[0016] As used herein, the terms "antigen-binding site" and "antigen-binding domain" can be used interchangeably and refer to the region of an antibody molecule that actually binds to an antigen. Preferably, the antigen-binding site of an antibody molecule used in the present invention comprises a VH / VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH), said VH / VL pair may be comprised in a single polypeptide chain or in two isolated polypeptide chains. In a preferred embodiment, the antibody molecule of the present invention comprises at least one antigen-binding site that specifically binds to GPRC5D, at least one antigen-binding site that specifically binds to BCMA, and at least one antigen-binding site that specifically binds to CD3. In one embodiment, the antibody of the present invention is a trivalent trispecific antibody, a tetravalent trispecific antibody, or a hexavalent trispecific antibody.

[0017] As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or a different epitope of a different antigen. The antibody provided herein is a trispecific antibody against GPRC5D, BCMA and CD3.

[0018] The term "immunoglobulin molecule" refers to a protein having the structure of a naturally occurring antibody. For example, the IgG class of immunoglobulins is a heterotetrameric glycoprotein of about 150,000 daltons consisting of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has one heavy chain variable region (VH), also called the heavy chain variable domain, followed by three heavy chain constant domains (CH1, CH2 and CH3). Similarly, from the N-terminus to the C-terminus, each immunoglobulin light chain has one light chain variable region (VL), also called the light chain variable domain, followed by one light chain constant domain (CL). The heavy chains of immunoglobulins can be classified into one of five classes called α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), where some classes can be further divided into subclasses, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chains of immunoglobulins can be classified into one of two types, called κ and λ, depending on the amino acid sequence of their constant domain. IgG immunoglobulins are essentially composed of two Fab molecules connected via an immunoglobulin hinge region and two dimerized Fc regions.

[0019] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of heavy and light chains of natural antibodies usually have a similar structure, where each domain contains four conserved framework regions (FR) and three complementarity determining regions. In some circumstances, a single VH or VL domain can be sufficient to confer antigen binding specificity.

[0020] "Complementarity determining regions" or "CDR regions" or "CDRs" or "hypervariable regions" are the regions in an antibody variable domain that are hypervariable in sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigen epitopes. The CDRs of heavy and light chains are numbered sequentially from the N-terminus and are usually referred to as CDR1, CDR2 and CDR3. The CDRs located in the heavy chain variable domain of an antibody are also referred to as HCDR1, HCDR2 and HCDR3, and the CDRs located in the light chain variable domain of an antibody are referred to as LCDR1, LCDR2 and LCDR3. For a given amino acid sequence of a light chain variable region or heavy chain variable region, the CDR sequence can be determined by various methods known in the art, such as Chothia, which is based on the three-dimensional structure of the antibody and the topology of the CDR loop, Kabat, which is based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (International Immunogenetics Information System, World Wide Web imgt.cines.fr / ), and the North CDR definition, which is based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

[0021] For example, different definitions of the CDR regions as numbered by Kabat and Chothia are used. [Table 1]

[0022] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" includes a CDR sequence determined by any one of the above methods.

[0023] CDRs can also be determined based on having the same Kabat numbering position as the reference CDR sequence. Unless otherwise specified, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0024] The term "Fc domain" or "Fc region" is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains, namely a CH2 domain, a CH3 domain and an optional CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain contains the second and third constant domains (CH2 domain and CH3 domain) from the two heavy chains of IgG, IgA and IgD class antibodies, or the second, third and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) from the two heavy chains of IgM and IgE class antibodies. In this specification, unless otherwise specified, the numbering of amino acid residues in the Fc region or heavy chain constant region is based on the EU numbering system (also called the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0025] The term "effector function" refers to a biological activity associated with the immunoglobulin Fc region that varies with immunoglobulin isotype. Examples of immunoglobulin effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors) and B cell activation.

[0026] The term "chimeric antibody" refers to an antibody molecule in which (a) the constant region or a portion thereof has been altered, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered type, effector function and / or species or an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug) that confers new properties to the chimeric antibody, or (b) the variable region or a portion thereof has been altered, replaced or exchanged by a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by changing its constant region to a constant region derived from a human immunoglobulin. Due to the alteration to a human constant region, the chimeric antibody retains its specificity for antigen recognition and has reduced antigenicity in humans compared to the original mouse antibody.

[0027] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) and is less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and modifying the remaining parts of the antibody to their corresponding human-derived parts (i.e., the parts of the constant and variable regions that are not involved in binding are the corresponding parts of a human antibody). See, for example, Padlan, Anatomy of the antibody molecule, Mol. Immun., 1994, 31:169-217. Other examples of human antibody engineering techniques include, but are not limited to, the Xoma technology disclosed in US 5,766,886.

[0028] As used herein, the terms "binding" or "specifically binding" mean that the binding action is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The binding ability of an antigen-binding site for a particular antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other conventional binding assays known in the art.

[0029] "Affinity" or "binding affinity" refers to the affinity of intrinsic binding, which reflects the interaction between members of a binding pair. The affinity of a molecule X for a partner Y can usually be expressed as a dissociation constant (KD), which is the ratio of the dissociation rate constant and the association rate constant (kdis and kkon, respectively). Affinity can be measured by conventional methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay described herein.

[0030] "Percentage identity" of an amino acid sequence refers to the percentage of amino acid residues in a candidate sequence that match those in the specific amino acid sequence set forth herein, comparing the candidate sequence to the specific amino acid sequence set forth herein, and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and where any conservative substitutions are not considered part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention, which have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or more identity, to the antibody molecules and sequences specifically disclosed herein. The variants may include conservative modifications. In the case of a polypeptide sequence, "conservative modifications" include substitutions, deletions, or additions to a polypeptide sequence, whereby a particular amino acid is replaced with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the invention. Below are eight pairs of amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G), 2) Aspartic acid (D), Glutamic acid (E), 3) Asparagine (N), Glutamine (Q), 4) Arginine (R), Lysine (K), 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V), 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W), 7) Serine (S), Threonine (T), and 8) Cysteine ​​(C), Methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the term "conservative sequence modifications" refers to amino acid modifications that have no appreciable effect on or do not alter the binding characteristics of an antibody containing the amino acid sequence.

[0031] The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells", which include the originally transformed cell and their derived progeny. Host cells may be any type of cell line used to produce the antibody molecules of the invention, and include eukaryotic cells, such as mammalian cells, insect cells, yeast cells, and prokaryotic cells, such as E. coli cells. Host cells include cultured cells, including cells within transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0032] The term "expression vector" refers to a vector containing a recombinant polynucleotide, including an expression control sequence operatively linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., nude or contained in liposomes), viruses (e.g., lentiviruses, retroviruses, adenoviruses, adeno-associated viruses) into which the recombinant polynucleotide has been incorporated.

[0033] Mammals include, but are not limited to, domestic animals (e.g., dairy cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats). In particular, the subject refers to a human.

[0034] The term "treatment" refers to a clinical intervention to alter the natural course of a disease in a subject being treated. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or alleviation of the disease state, and alleviation or improvement of prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the development of disease or the progression of disease.

[0035] The terms "tumor" and "cancer" are used interchangeably herein and include solid and liquid tumors.

[0036] The term "GPRC5D" refers to tumor-associated antigen G protein-coupled receptor family C group 5 member D (e.g., human GPRC5D protein with accession number UniProt Q9NZD1). As used herein, "antigen-binding specificity for GPRC5D" refers to an antibody or antibody fragment, e.g., scFv or Fab, that specifically binds to GPRC5D. In one embodiment, the antigen-binding site of the antibody molecule of the present invention that binds to GPRC5D may have high affinity binding activity, e.g., an EC50 value of 1 nM to 100 nM, e.g., 20 nM to 60 nM, for cells expressing GPRC5D, as detected by flow cytometry. In one embodiment, the antigen-binding specificity has cross-reactivity to human and monkey GPRC5D.

[0037] The term "BCMA" refers to the tumor associated antigen B cell maturation antigen (e.g., human BCMA protein with accession number UniProt Q02223), also called BCMA, TR17_human, TNFRSF17. As used herein, "antigen binding specificity for BCMA" refers to an antibody or antibody fragment, e.g., scFv or Fab, that specifically binds to BCMA. In one embodiment, the antigen binding site of the antibody molecule of the present invention that binds to BCMA may have a high affinity binding activity for BCMA, e.g., a KD value of 0.1 nM to 10 nM, e.g., 0.1 nM to 5 nM, as detected by biofilm layer optical interference techniques. In one embodiment, the antigen binding specificity has cross-reactivity to human and monkey BCMA.

[0038] The term "CD3" refers to the T cell engaging antigen T cell surface glycoprotein CD3 (e.g., human CD3 protein with accession number UniProt P07766). As used herein, "antigen binding specificity for CD3" refers to an antibody or antibody fragment, e.g., scFv or Fab, that specifically binds to CD3. In one embodiment, the antigen binding site of the antibody molecule of the present invention that binds to CD3 may have a high affinity binding activity for the CD3 epsilon chain, e.g., a KD value of 1 nM to 50 nM, e.g., 0.1 nM to 5 nM, as detected by biofilm layer light interference techniques. Preferably, a high affinity CD3 antigen binding site with a KD value of less than 50 nM is used in the trispecific antibody molecule of the present invention. In one embodiment, the antigen binding specificity has cross-reactivity to human and monkey CD3.

[0039] When describing an antibody structure of the present invention, the term "N-terminus" refers to the last amino acid at the N-terminus and the term "C-terminus" refers to the last amino acid at the C-terminus.

[0040] "Knob-in-hole" mutation or "knob-in-hole" mutation, as used herein, refers to the introduction of mutations into a first Fc polypeptide and a second Fc polypeptide by the "knob-in-hole" technique to form a protrusion ("knob") and a complementary cavity ("hole") at the interface of the first Fc polypeptide and the interface of the second Fc polypeptide, respectively. As known in the art, the "knob-in-hole" technique can facilitate accurate binding of each chain of an antibody molecule by modifying the interface between different chains of the antibody molecule. In general, the technique involves introducing a "protrusion" at the interface of one chain and a corresponding "cavity" at the interface of the other chain that is paired with it, such that the protrusion can be positioned in the cavity. One preferred interface includes the CH3 domain of the heavy chain constant domain of one chain and the CH3 domain of the heavy chain constant domain of the other chain that is paired with it. A protuberance can be constructed by replacing small amino acid side chains at the interface of the CH3 domain of the heavy chain constant domain from one chain with relatively large side chains (e.g., tyrosine or tryptophan). A compensatory cavity of the same or similar size as the protuberance is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other paired chain by replacing large amino acid side chains with relatively small side chains (e.g., alanine or threonine). Another selectable interface includes the CL domain of the light chain and the CH1 domain of the heavy chain, as in the Fab fragment, which promotes correct heterodimerization between the two chains of the Fab fragment by establishing a protuberance-cavity interaction.

[0041] "Single-chain variable fragment" or "scFv" is used herein to refer to a single-chain antibody fragment comprising a heavy chain variable domain VH and a light chain variable domain VL linked by a linker, where VH and VL pair to form an antigen-binding site. "Disulfide bond stabilized single-chain variable fragment" or "dsscFv" is used herein to refer to a disulfide bond stabilized single-chain variable fragment, where the scFv fragment forms a disulfide bond linkage between the VH and VL domains by artificially introducing cysteine ​​mutations into the VH and VL domains.

[0042] "Fv fragment" is used herein to refer to an antibody fragment comprising a heavy chain variable domain, VH, and a light chain variable domain, VL. "Disulfide bond stabilized variable fragment" or "dsFv" is used herein to refer to an Fv fragment stabilized by an artificially introduced disulfide bond between the VH and VL domains.

[0043] "Single domain antibody" or "sdAb" is used herein to refer to an antibody fragment consisting of a single variable antibody domain, e.g., VH or VL, such as a heavy chain variable domain from a camelid heavy chain antibody, or a VH-like single domain (v-NAR) from a fish IgNAR. The single variable domain of a single domain antibody does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include single domain antibodies from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks).

[0044] "Fab fragment" or "Fab" is used herein to refer to an immunoglobulin fragment comprising two polypeptide chains, the immunoglobulin heavy chain variable domain VH, the heavy chain constant domain CH1, the light chain variable domain VL and the light chain constant domain CL, wherein one polypeptide chain comprises, from the N-terminus to the C-terminus, VH and one constant domain selected from CH1 and CL, and the other polypeptide chain comprises, from the N-terminus to the C-terminus, VL and the other constant domain selected from CL and CH1, wherein the VH and VL domains pair to form an antigen-binding site. Herein, when one polypeptide chain of a Fab comprises a VH linked to CL and the other polypeptide chain comprises a VL linked to CH1, the Fab is also referred to as a crossFab.

[0045] The terms "single-chain Fab" or "scFab" are used herein to refer to a single-chain polypeptide formed by joining the two chains of Fab fragments with a linker.

[0046] In some embodiments of the invention, the antibody of the invention may comprise a Fab fragment selected from (i) a Fab consisting of one chain comprising VH-CH1 and one chain comprising VL-CL, and (ii) a crossFab consisting of one chain comprising VH-CL and one chain comprising VL-CH1. In other embodiments of the invention, the antibody of the invention may comprise a single-chain Fab fragment selected from (i) a single-chain Fab consisting of VH-CH1-linker-VL-CL, and (ii) a single-chain Fab consisting of VH-CL-linker-VL-CH1.

[0047] In the present specification, the immunoglobulin constant domain can be selected based on the expected function of the antibody molecule.For example, the constant domain can be an immunoglobulin constant domain of human IgG, such as an IgA, IgD, IgE, IgG or IgM domain, particularly a constant domain of human IgG1, IgG2, IgG3 or IgG4, and preferably a constant domain of human IgG1.As an example, the Fab fragment of the antibody can include the CH and CL constant domains derived from IgG1.As a further example, the Fc region of the antibody can include the CH2 and CH3 domains derived from IgG1.

[0048] As used herein, the term "flexible connecting peptide", "connecting peptide" or "linker" refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or a hinge region derived from an immunoglobulin. In one embodiment, the connecting peptide has a length of 5 to 50 amino acids, such as 10, 15, 20, 25 or 30 amino acids. In one embodiment, the connecting peptide comprises the amino acid sequence (G4S)n, where n is an integer equal to or greater than 1, e.g., n is an integer equal to or greater than 2, 3, 4, 5, 6 or 7. In one embodiment, the connecting peptide comprises the amino acid sequence TS(G4S)n, where n is an integer equal to or greater than 1, e.g., n is an integer equal to or greater than 2, 3, 4, 5, 6 or 7. In a further embodiment, the connecting peptide is a hinge region derived from an immunoglobulin, for example, an amino acid sequence of a hinge region containing "CPPC", for example, the amino acid sequence "EPKSCDKTHTCPPCP" (SEQ ID NO: 114) or "EPKSSDKTHTCPPCP" (SEQ ID NO: 115). The connecting peptide linking each domain of the antibody molecule applicable to the present invention may be, for example, GGG (SEQ ID NO: 116), DGGGS (SEQ ID NO: 117), TGEKP (SEQ ID NO: 118), GGRR (SEQ ID NO: 119), EGKSSGSGSESKVD (SEQ ID NO: 120), KESGSVSSEQLAQFRSLD (SEQ ID NO: 121), GGRRGGGS (SEQ ID NO: 122), LQRDGERP (SEQ ID NO: 123), LRQKDGGGSERP (SEQ ID NO: 124), and GSTSGSGKPGSGEGSTKG (SEQ ID NO: 125), but is not limited thereto. Alternatively, computer programs can be used to mimic the three-dimensional structures of proteins and peptides, or suitable flexible connecting peptides can be rationally designed by phage display methods.

[0049] I. Trispecific antibody molecules of the invention The trispecific antibody molecule of the present invention is a Y-shaped antibody molecule. In this specification, the term "Y-shaped antibody molecule" refers to an antibody molecule with a Y-shaped structure including two antibody arms and one stem, where the two antibody arms are connected to the stem of the Y-shaped structure formed by pairing with two antibody Fc domains to form two branches of the Y-shaped structure. One typical Y-shaped antibody molecule is an immunoglobulin IgG molecule. The Y-shaped structure of an immunoglobulin IgG molecule is composed of three regions, two antibody arms (Fab) and one stem (Fc), where a flexible hinge region (Hinge) connects the antibody stem (Fc) and the antibody arm (Fab). In an IgG molecule, the two arms are used for specific binding to an antigen, while the stem determines the type and functional properties of the antibody. In this specification, the Y-shaped antibody molecule also covers molecules having the same or similar Y-shaped structure as an IgG molecule, such as Y-shaped molecules having a complex component in the antibody arm and / or stem of the antibody. Preferably, the antibody molecule of the present invention is a trispecific antibody molecule having multiple moieties.

[0050] In the Y-shaped antibody molecule of the present invention, the antibody arms can be composed of antigen-binding domains such as Fab, scFv, sdAB, etc., and the antigen-binding domains constituting the two antibody arms can target homologous or different antigens and / or epitopes and have homologous or different binding specificities. In the Y-shaped antibody molecule of the present invention, the stem of the antibody molecule can be composed of two paired immunoglobulin Fc domains, where the Fc domains may or may not independently contain mutations. When the antibody molecule is an asymmetric IgG-like molecule, preferably the two Fc domains in the stem contain mutations that promote their pairing and dimerization, such as complementary "knobs-in-holes" mutations. Furthermore, the Fc domain of the stem may contain other mutations, such as cysteine ​​mutations (facilitating the formation of disulfide bond linkages between the first and second Fc), charge pairing mutations, and / or mutations that reduce or eliminate effector function (e.g., mutations that reduce or eliminate ADCC activity, such as the combination of L234A / L235A mutations). The two antibody arms of the Y-shaped antibody molecule of the present invention may each be covalently linked to the respective Fc domain of the stem directly or via a linking peptide, and preferably the arms are linked to the Fc domain of the stem via the hinge region of an immunoglobulin.

[0051] In addition to the arms and stem, the Y-shaped antibody molecule of the present invention preferably further comprises a conjugated moiety conjugated to the arms and / or stem, said conjugated moiety comprising an additional antigen-binding domain, preferably an scFv, such as Fab, scFv and sdAB. The antigen-binding domain in the conjugated moiety can confer a different antigen-binding specificity than the antibody arms. For example, the two antibody arms of the Y-shaped molecule may comprise antigen-binding domains providing a first and a second antigen-binding specificity, respectively, and the conjugated moiety of the Y-shaped molecule comprises an antigen-binding domain providing a third antigen-binding specificity, and the first, second and third antigen-binding specificities are different from each other. Alternatively, the two antibody arms of the Y-shaped molecule may comprise antigen-binding domains providing a first antigen-binding specificity, and the conjugated moiety of the Y-shaped molecule comprises antigen-binding domains providing a second and a third antigen-binding specificity, and the first, second and third antigen-binding specificities are different from each other.

[0052] In the Y-shaped antibody molecule of the present invention, the first, second and third antigen binding specificities are binding specificities for different antigens selected from CD3, BCMA and GPRC5D, respectively. For example, the first and second antigen binding specificities may be for different tumor-associated antigens (TAA) selected from GPRC5D and BCMA, respectively, and the third antigen binding specificity may be for T-cell surface antigen (TEA) CD3. Alternatively, the first (or second) and third antigen binding specificities may be for different tumor-associated antigens (TAA) selected from GPRC5D and BCMA, respectively, and the second (or first) antigen binding specificity may be for T-cell surface antigen (TEA).

[0053] Depending on the structure of the arm, stem and conjugated moiety, the Y-shaped antibody molecule of the invention may comprise at least two polypeptide chains, e.g., two or four polypeptide chains. Herein, a polypeptide chain comprising a stem Fc domain is referred to as a heavy chain, whereas a polypeptide chain not comprising an Fc domain is referred to as a light chain. Thus, in some embodiments, the Y-shaped molecule of the invention is a di-chain molecule comprising a first heavy polypeptide chain and a second heavy polypeptide chain, while in some other embodiments, the Y-shaped molecule of the invention is a quadri-chain molecule comprising two heavy polypeptide chains and two light polypeptide chains. In the Y-shaped molecule of the invention comprising a conjugated moiety, the conjugated moiety may be conjugated to a heavy polypeptide chain of the antibody molecule (e.g., to the C-terminus of the Fc domain of the stem) or to a light polypeptide chain of the antibody molecule (e.g., to the C-terminus of the Fab light chain of an antibody arm comprising a Fab antigen-binding site). Thus, for example, an antibody of the present invention may comprise two heavy polypeptide chains and two light polypeptide chains, where a first heavy chain and a first light chain pair to form one antibody arm of the antibody, a second heavy chain and a second light chain pair to form another antibody arm of the antibody, and an Fc domain of the first heavy chain and an Fc domain of the second heavy chain pair to form an antibody stem, where the C-terminus of the first heavy chain or the second heavy chain further comprises a single chain antibody fragment (such as an scFv) that forms an antibody complex, or where the C-terminus of the first light chain and the second light chain further comprises a single chain antibody fragment (such as an scFv) that forms an antibody complex.

[0054] In one embodiment, the present invention therefore provides a trispecific Y-shaped antibody molecule. The Y-shaped antibody molecule of the present invention has the following formula: (M1:M2-(X1)p)-Fc::Fc-(X2)q (Formula I) wherein: M1 and M2 respectively represent the first and second antibody arms of a Y-shaped antibody molecule comprising an antigen-binding site, where the antigen-binding sites of the first and second antibody arms may be homologous or different; Fc::Fc represents the stem of a Y-shaped antibody molecule located at the C-terminus of the first and second antibody arms and is composed of paired and dimerized first and second Fc domains; X1 represents a conjugated moiety conjugated to the C-terminus of an antibody arm, and X2 represents a conjugated moiety conjugated to the C-terminus of the stem, wherein the conjugated moiety comprises an antigen-binding site, wherein the conjugated moiety is conjugated to an antibody molecule directly or via a linking peptide, and In the formula, p and q each represent an integer of 0, 1, or 2, and p and q are not 0 at the same time; wherein the antibody specifically binds to GPRC5D, BCMA and CD3 via an antibody arm and a conjugation moiety; Preferably, any one of p and q is 0 when the first antibody arm and the second antibody arm bind different antigens, where the antibody arms M1 and M2 provide the first and second antigen-binding specificities, and the composite component provides a third antigen-binding specificity; or Preferably, p and q are not both 0 when the first and second antibody arms bind to the same antigen, where antibody arms M1 and M2 provide a first antigen-binding specificity and conjugate moieties X1 and X2 provide a second and third antigen-binding specificity, respectively.

[0055] In some embodiments, the antibody arms (M1 and M2) of the antibody molecule comprise an antigen binding site selected from a Fab or scFab (preferably a Fab), and any one of p and q is 0, wherein the antibody arms M1 and M2 bind to a first and a second antigen, respectively, and the composite moiety (X1 or X2) binds to a third antigen, wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3; Preferably, when p=0, q=1 and the conjugation moiety X2 is conjugated to one of the two Fc domains of the stem of the antibody molecule, or Preferably, when q=0, p=2 and conjugation moiety X1 is conjugated to the Fab light chain of both antibody arms M1 and M2 of the antibody molecule.

[0056] In some embodiments, the antibody arms (M1 and M2) of the antibody molecule comprise an antigen binding site selected from a Fab or scFab (preferably Fab), and p and q are not both 0, wherein the antibody arms M1 and M2 bind to the same first antigen, and the complex moieties X1 and X2 bind to a second antigen and a third antigen, respectively, wherein the first, second and third antigens are different from each other and are independently selected from GPRC5D, BCMA and CD3; Preferably, p=2 and q=2, where conjugation moiety X1 is conjugated to the Fab light chains of both antibody arms M1 and M2 of the antibody molecule, and conjugation moiety X2 is conjugated to the two Fc domains of the stem of the antibody molecule. In a further embodiment, the antibody arms (M1 and M2) of the antibody molecule comprise an antigen binding site selected from an scFv (preferably a dsscFv) and p=0, q=1 or 2, wherein the antibody arms M1 and M2 are bound to a first and a second antigen, respectively, and the conjugate moiety X2 is bound to a third antigen, wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3; Preferably, when p=0, q=1 and the conjugation moiety X2 is conjugated to the C-terminus of any one of the two Fc domains in the stem of the antibody molecule.

[0057] In any one of the above embodiments, preferably the conjugate component comprises an antigen binding site selected from an scFv, a dsFv and a dsAb, preferably an scFv, more preferably a dsscFv. In any one of the above embodiments, preferably the first and second Fc domains comprise a hinge region having "CPPC" amino acid residues, thereby allowing the antibody molecule of the invention to form an interchain disulfide bond between the first and second Fc domains to promote correct pairing of the polypeptide chains of the antibody molecule of the invention. Preferably, in the antibody molecule of the invention, the antibody arm is fused to the N-terminus of the Fc region by the hinge region of the Fc region. In any one of the above embodiments, preferably the conjugated moiety is conjugated to the stem and / or arm of the antibody molecule by a flexible connecting peptide. Preferably, the length of said connecting peptide is 5-15 amino acids long, for example 10, 12, 15 amino acids long. Preferably, said connecting peptide comprises the amino acid sequence TS(G4S)n or (G4S)n, where n=1, 2 or 3, preferably n=2.

[0058] Thus, in some preferred embodiments, when any one of p and q in formula I is 0, the present invention provides a compound of the formula: (M1:M2-(X1)p)-Fc::Fc-(X2)q (Formula I) and providing an IgG-like antibody molecule having the structure: M1 and M2 comprise Fab or scFab, preferably Fab, that bind the first and second antigens, respectively; where p=0, X2 comprises an scFv that binds a third antigen; or When q=0, X1 comprises an scFv that binds to a third antigen; wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3. In some further preferred embodiments, when p=0 in formula I, the present invention provides a compound of the formula: (M1:M2)-Fc::Fc-(X2)q, (Format_2) and providing an IgG-like antibody molecule having the structure: q=1, M1 and M2 comprise Fab or scFab, preferably Fab, that bind the first and second antigens, respectively; and X2 comprises an scFv that binds to a third antigen, wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0059] In some further preferred embodiments, when q=0 in formula I, the present invention provides a compound of the formula: (M1:M2-(X1)p)-Fc::Fc, (Format_7) and providing an IgG-like antibody molecule having the structure: p=2, M1 and M2 comprise Fabs that bind to the first and second antigens, respectively; and X2 comprises an scFv that binds to a third antigen, wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0060] In some further embodiments, when p=2 and q=2 in formula I, the present invention provides a compound of the formula: (M1:M2-(X1)p)-Fc::Fc-(X2)q, (Format_6) and providing an IgG-like antibody molecule having the structure: M1 and M2 comprise a Fab or scFab, preferably a Fab, that binds to the same first antigen, and X1 and X2 comprise scFvs that bind to a second antigen and a third antigen, respectively; wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0061] In some further embodiments, when p=0 and q=1 in formula I, the present invention provides a compound of the formula: (M1:M2)-Fc::Fc-X2, (Format_1) and providing an IgG-like antibody molecule having the structure: M1, M2 and X2 comprise scFvs that bind to the first, second and third antigens, respectively; wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0062] The antibody arms, conjugate moieties and stems constituting the trispecific antibody molecule of the present invention are described in detail below. As will be apparent to those skilled in the art, these descriptions apply to any of the above-described trispecific antibody molecules of the present invention, such as the molecules of formula I and the molecules having the structures of Format_1, 2, 6 and 7.

[0063] Antibody Arms and Conjugate Components of the Antibody Molecule of the Invention The antibody molecule of the present invention provides an antigen binding site that specifically binds to a first, second and third antigen by way of the antibody arms and the antigen binding site of the composite moiety, wherein the first, second and third antigens are different from each other and independently selected from the tumor associated antigen GPRC5D, the tumor associated antigen BCMA and the T cell engaging antigen CD3.

[0064] In one embodiment, the invention provides an antibody molecule of Formula I, where when p=0 or q=0, the first antibody arm and the second antibody arm of the antibody provide the first and second antigen-binding sites, respectively, and the composite moiety provides the third antigen-binding site.

[0065] In another embodiment, the invention provides an antibody molecule of Formula I, where when p and q are not both 0, the first and second antibody arms of the antibody provide a first antigen-binding site, and a conjugated moiety conjugated to the C-terminus of the antibody arms provides a second antigen-binding site, and a conjugated moiety conjugated to the antibody stem provides a third antigen-binding site.

[0066] The antigen-binding site of the trispecific antibody molecule applicable to the present invention (including the antigen-binding site that specifically binds to GPRC5D, BCMA and CD3) may comprise any antibody or antibody fragment capable of binding to the target antigen. For example, in the antibody molecule of formula I of the present invention, the antigen-binding site in the antibody arm and the composite component may be independently scFv, dsFv, Fab, scFab or sdAb. Preferably, in some embodiments, the antigen-binding site of the antibody arm is selected from Fab and scFab, preferably Fab. Preferably, in some other embodiments, the antigen-binding site of the composite component is selected from scFv, dsFv and dsAb, preferably scFv, more preferably disulfide-bond stabilized scFv (i.e. dsscFv). In some more preferred embodiments, the antigen-binding site of the antibody arm is Fab and the antigen-binding site of the composite component is scFv, especially dsscFv.

[0067] Herein, Fab_GPRC5D, Fab_BCMA and Fab_CD3 are used to represent the antigen-binding sites of the Fab format that bind GPRC5D, BCMA and CD3, respectively. Herein, ScFv_GPRC5D, ScFv_BCMA and ScFv_CD3 are used to represent the antigen-binding sites of the scFv format that bind GPRC5D, BCMA and CD3, respectively.

[0068] In some embodiments, the Fab antigen-binding site in the antibody molecule of the invention is composed of two polypeptide chains comprising immunoglobulin VH, CH1, VL and CL domains, where the VH and VL pair and the CH1 and CL pair to form the antigen-binding site. In some embodiments, in the Fab, one chain comprises VH and CH1 (i.e., VH-CH1) from N-terminus to C-terminus, and the other chain comprises VL and CL (i.e., VL-CL) from N-terminus to C-terminus. In some other embodiments, in the Fab, one chain comprises VH and CL (i.e., VH-CL) from N-terminus to C-terminus, and the other chain comprises VL and CH1 (i.e., VL-CH1) from N-terminus to C-terminus. In some embodiments, the Fab constitutes the antigen-binding site of the antibody arm of the antibody molecule of the invention. In said embodiment, the Fab can be fused to the N-terminus of the Fc domain of the antibody stem by the C-terminus of the chain comprising the VH, or alternatively, wherein the Fab is fused to the N-terminus of the Fc domain of the antibody stem by the C-terminus of the chain comprising the VL. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain and is attached to the Fc region of the antibody stem by the C-terminus of the VH-CH1 chain. Preferably, when the antibody molecule of the invention comprises a conjugation moiety conjugated to an antibody arm, the conjugation moiety is conjugated to the C-terminus of a Fab chain that is not linked to the antibody stem. Herein, a Fab chain that is not linked to an antibody stem is also referred to as a light chain of the Fab. Thus, in some embodiments, the antibody molecule of the invention has a conjugation moiety conjugated to a Fab light chain of an antibody arm.

[0069] In some other embodiments, the scFab antigen-binding site in the antibody molecule of the invention is composed of a single polypeptide chain comprising immunoglobulin VH, CH1, VL and CL domains, where the VH and VL pair and the CH1 and CL pair to form the antigen-binding site. In some embodiments, the scFab comprises, from N-terminus to C-terminus, VH-CH1, a linker, and VL-CL. In some other embodiments, the scFab comprises, from N-terminus to C-terminus, VL-CL, a linker, and VH-CH1. In some further embodiments, the scFab comprises, from N-terminus to C-terminus, VH-CL, a linker, and VL-CH1. In some other embodiments, the scFab comprises, from N-terminus to C-terminus, VL-CH1, a linker, and VH-CL. In some embodiments, the scFab comprises, from N-terminus to C-terminus, VL-CH1, a linker, and VH-CL. In some embodiments, the scFab constitutes the antigen-binding site of an antibody arm of the antibody molecule of the invention. In some embodiments, the scFab is fused by the C-terminus of its polypeptide chain to the N-terminus of the Fc domain of the antibody stem. Preferably, the scFab comprises, from N-terminus to C-terminus, VL-CL, a linker, and VH-CH1, and is linked to the Fc region of the antibody stem by the C-terminus of the CH1 domain. Preferably, when an antibody molecule of the invention comprises an scFab antibody arm, the conjugated component of the antibody molecule is conjugated only to the C-terminus of the Fc domain of the antibody stem.

[0070] In some embodiments, the Fab or scFab comprised in the antibody molecule of the invention comprises a CH1 domain derived from an IgG immunoglobulin, such as the CH1 domain of IgG1, preferably the CH1 domain of human IgG1. In some preferred embodiments, the CH1 domain comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity thereto. In some embodiments, the Fab or scFab comprised in the antibody molecule of the invention comprises a kappa light chain constant domain derived from an IgG immunoglobulin, such as the CL of IgG1. κ Domain, preferably human IgG1 CL κFor example, the Fab or scFab comprises a kappa light chain constant domain having an amino acid sequence of SEQ ID NO: 105, or an amino acid sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity thereto. In some other embodiments, the Fab or scFab comprised in the antibody molecule of the invention comprises a lamda light chain constant domain derived from an IgG immunoglobulin, such as the CL domain of IgG1. λ Domain, preferably human IgG1 CL λ For example, the Fab comprises a lamda light chain constant domain having an amino acid sequence of SEQ ID NO: 106, or an amino acid sequence having at least 90%, 95%, 97%, 98% or 99% sequence identity thereto. In some embodiments, when antibody arms M1 and M2 of the antibody molecule of the present invention comprise Fabs or scFabs (preferably Fabs) that bind to different antigens, the light chain constant domains of the Fabs or scFabs of antibody arm M1 and antibody arm M2 are different from each other. For example, in some embodiments, the Fab of antibody arm M1 comprises a kappa light chain constant domain and the Fab of antibody arm M2 comprises a lamda light chain variable domain, or the Fab of antibody arm M1 comprises a lamda light chain constant domain and the Fab of antibody arm M2 comprises a kappa light chain variable domain.

[0071] In some embodiments, the scFv antigen-binding site contained in the antibody molecule of the present invention is composed of a single polypeptide chain comprising immunoglobulin VH and VL domains, wherein the VH and VL are linked by a linker and paired to form the antigen-binding site. In some embodiments, the scFv is in a trans configuration and comprises, from N-terminus to C-terminus, a VH, a linker, and a VL (VH-linker-VL). In some other embodiments, the scFv is in a cis configuration and comprises, from N-terminus to C-terminus, a VL, a linker, and a VH (VH-linker-VL). In some embodiments, the linker is a peptide linker composed of amino acid residues. Suitable peptide linkers are well known to those skilled in the art. In one embodiment, the linker is 10 to 50 amino acids long, for example 5 to 30 amino acids long, for example 15 amino acids or 20 amino acids long. In one embodiment, the linker comprises the amino acid sequence (G4S)n, where n=1, 2, 3, 4 or 5, preferably n=3 or 4, more preferably n=4. In some embodiments, an scFv constitutes the antigen-binding site of the antibody arm of an antibody molecule of the invention and is fused by the C-terminus of its polypeptide chain to the N-terminus of the Fc domain of the antibody stem.

[0072] In some other embodiments, an scFv constitutes the antigen-binding site of the composite component of the antibody molecule of the invention. In some embodiments, said scFv is fused by the N-terminus of its polypeptide chain to the C-terminus of the antibody stem and / or to the C-terminus of the antibody arm. Preferably, in the antibody molecule of the invention, the antibody arm comprises a Fab and the composite component comprises an scFv, wherein said scFv is fused by the N-terminus of its polypeptide chain to the C-terminus of the antibody stem Fc and / or to the C-terminus of the antibody arm Fab light chain. In some preferred embodiments, the scFv antigen-binding site contained in the antibody molecule of the present invention is a disulfide bond stabilized scFv, i.e., dsscFv. When the antigen-binding site is a dsscFv, the disulfide bond introduced between the VH and VL variable domains mutated into the scFv results in the following residue pairs (the positions below are determined by Kabat numbering): It can be located between residue 37 of VH + residue 95 of VL, residue 44 of VH + residue 100 of VL, residue 44 of VH + residue 105 of VL, residue 45 of VH + residue 87 of VL, residue 55 of VH + residue 101 of VL, residue 100 of VH + residue 50 of VL, residue 100b of VH + residue 49 of VL, residue 98 of VH + residue 46 of VL, residue 105 of VH + residue 43 of VL, and residue 106 of VH + residue 57 of VL. Preferably, to form a dsscFv, cysteine ​​substitutions are introduced at positions 44 of VH and 100 of VL, so that a disulfide bond can be formed between the two residues when the VH and VL are paired.

[0073] Antigen-binding site of GPRC5D The GPRC5D antigen-binding site of the antibody molecule applicable to the present invention may be any antibody or antibody fragment capable of binding to GPRC5D. For example, in the antibody molecule of the present invention, the GPRC5D antigen-binding site contained in the antibody arm or composite component may be independently scFv, dsFv, Fab, scFab or sdAb, and is preferably an antigen-binding site having the above-mentioned scFv or Fab structure. Preferably, the GPRC5D antigen-binding site of the antibody arm is selected from Fab and scFab, preferably Fab. Preferably, the GPRC5D antigen-binding site of the composite component is selected from scFv, dsFv and dsAb, preferably scFv, more preferably disulfide-bond-stabilized scFv (i.e., dsscFv). In one more preferred embodiment, the antibody comprises an antibody arm that binds to GPRC5D and the GPRC5D antigen-binding site of the antibody arm is Fab, and in another more preferred embodiment, the antibody comprises a composite component that binds to GPRC5D and the GPRC5D antigen-binding site of the composite component is an scFv, particularly a dsscFv.

[0074] In some preferred embodiments, the antigen-binding site that binds to GPRC5D, for example a GPRC5D antigen-binding site having the scFv or Fab structure described above, is (i) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 25 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 26, or (ii) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 27 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 28, or (iii) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 29 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 30; or (iv) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 31 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 32; or (v) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 98 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 99; or (vi) comprises the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 100, and the sequences of LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 101.

[0075] In some preferred embodiments, an antigen binding site that binds to GPRC5D, for example a GPRC5D antigen binding site having the scFv or Fab structure described above, comprises the following: (i) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 1 to 6, respectively; or (ii) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 7 to 12, respectively; or (iii) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 13 to 18, respectively; or (iv) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 19 to 24, respectively; or (v) A combination of CDR sequences selected from the sequences of heavy chain variable domain HCDR1, 2 and 3 and the sequences of light chain variable domain LCDR1, 2 and 3 shown in SEQ ID NOs: 13, 110 and 15 to 18, respectively.

[0076] Alternatively, the antigen binding site, e.g., scFv or Fab, that binds to GPRC5D comprises a variant of one of the combinations of CDR sequences, wherein the variant comprises a total of at least one and not more than 5, 4, 3, 2 or 1 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in 1, 2, 3, 4, 5 or preferably 6 CDR regions, preferably with the heavy chain CDR3 remaining unchanged.

[0077] In some preferred embodiments, an antigen binding site that binds to GPRC5D, for example a GPRC5D antigen binding site having the scFv or Fab structure described above, comprises the following: (a) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (b) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (c) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (d) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (e) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 98, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; and (f) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 100, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0078] In some preferred embodiments, an antigen binding site that binds to GPRC5D, for example a GPRC5D antigen binding site having the scFv or Fab structure described above, comprises the following: (a) a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 26, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (b) a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 28, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (c) a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (d) a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (e) a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 99, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; and (f) a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 101, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0079] In some preferred embodiments, an antigen binding site that binds to GPRC5D, for example a GPRC5D antigen binding site having the scFv or Fab structure described above, comprises the following: (a) a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO:25, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO:26, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (b) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (c) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (d) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (e) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 98, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 99, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (f) a combination of VH and VL amino acid sequences selected from: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 100, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 101, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0080] In some preferred embodiments, an antigen binding site that binds to GPRC5D, for example a GPRC5D antigen binding site having the scFv or Fab structure described above, comprises the following: (i) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 26, or (ii) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 27, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28, or (iii) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 30; or (iv) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 32; (v) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 99; (vi) a combination of VH and VL sequences selected from a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 101. In some preferred embodiments, when the antigen-binding site that binds to GPRC5D is a disulfide-stabilized scFv, said scFv further comprises a cysteine ​​substitution at position 44 of the VH domain and a cysteine ​​substitution at position 100 of the VL domain.

[0081] Antigen-binding site that binds to BCMA The BCMA antigen-binding site of the antibody molecule applicable to the present invention may be any antibody or antibody fragment capable of binding to BCMA. For example, in the antibody molecule of the present invention, the BCMA antigen-binding site contained in the antibody arm or composite component may be independently an scFv, dsFv, Fab, scFab or sdAb, and is preferably an antigen-binding site having the scFv or Fab structure described above. Preferably, the BCMA antigen-binding site of the antibody arm is selected from Fab and scFab, preferably Fab. Preferably, the BCMA antigen-binding site of the composite component is selected from scFv, dsFv and dsAb, preferably scFv, more preferably disulfide-stabilized scFv (i.e. dsscFv). In one more preferred embodiment, the antibody comprises an antibody arm that binds to BCMA and the BCMA antigen-binding site of the antibody arm is a Fab, and in another more preferred embodiment, the antibody comprises a composite component that binds to BCMA and the BCMA antigen-binding site of the composite component is a scFv, in particular a dsscFv.

[0082] In some preferred embodiments, an antigen binding site that binds to BCMA, for example a BCMA antigen binding site having the scFv or Fab structures described above, comprises the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO:39, and the sequences of LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO:40.

[0083] In some preferred embodiments, an antigen binding site that binds to BCMA, for example a BCMA antigen binding site having the scFv or Fab structure described above, comprises the following: - HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 33, - an HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 34, - an HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 35, LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 36, - LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 37, - LCDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 38, Alternatively, said antigen binding site which binds to BCMA comprises a variant of one of said CDR sequence combinations, wherein said variant comprises a total of at least 1 and not more than 5, 4, 3, 2 or 1 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in 1, 2, 3, 4, 5 or preferably 6 CDR regions, preferably wherein the heavy chain CDR3 remains unchanged.

[0084] In some preferred embodiments, the antigen binding site that binds to BCMA, for example a BCMA antigen binding site having the scFv or Fab structure described above, comprises a heavy chain variable domain VH having the amino acid sequence set forth in SEQ ID NO: 39 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0085] In some preferred embodiments, the antigen binding site that binds to BCMA, for example a BCMA antigen binding site having the scFv or Fab structure described above, comprises a light chain variable domain VL having an amino acid sequence as set forth in SEQ ID NO: 40 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0086] In some preferred embodiments, an antigen binding site which binds to BCMA, for example a BCMA antigen binding site having an scFv or Fab structure as described above, comprises a heavy chain variable domain VH having the amino acid sequence set forth in SEQ ID NO: 39, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and comprises a light chain variable domain VL having the amino acid sequence set forth in SEQ ID NO: 40, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0087] Preferably, the antigen binding site that binds BCMA, for example a BCMA antigen binding site having the scFv or Fab structure described above, comprises a VH domain having the amino acid sequence set out in SEQ ID NO: 39 and a VL domain having the amino acid sequence set out in SEQ ID NO: 40. In some preferred embodiments, when the antigen binding site that binds BCMA is a disulfide bond stabilized scFv, said scFv further comprises a cysteine ​​substitution at position 44 in the VH domain and a cysteine ​​substitution at position 100 in the VL domain.

[0088] Antigen-binding site that binds to CD3 The CD3 antigen-binding site of the antibody molecule applicable to the present invention may be any antibody or antibody fragment capable of binding to CD3. For example, in the antibody molecule of the present invention, the CD3 antigen-binding site contained in the antibody arm or composite component may be independently scFv, dsFv, Fab, scFab or sdAb, and is preferably an antigen-binding site having the scFv or Fab structure described above. Preferably, the CD3 antigen-binding site of the antibody arm is selected from Fab and scFab, preferably Fab. Preferably, the CD3 antigen-binding site of the composite component is selected from scFv, dsFv and dsAb, preferably scFv, more preferably disulfide-bond-stabilized scFv (i.e. dsscFv). In one more preferred embodiment, the antibody comprises an antibody arm that binds to CD3 and the CD3 antigen-binding site of the antibody arm is a Fab, and in another more preferred embodiment, the antibody comprises a composite component that binds to CD3 and the CD3 antigen-binding site of the composite component is a scFv, in particular a dsscFv.

[0089] In some preferred embodiments, an antigen binding site that binds to CD3, e.g., a CD3 antigen binding site having the scFv or Fab structure described above, comprises the sequences of HCDR1, 2 and 3 of the heavy chain variable domain set forth in SEQ ID NO:48, and the sequences of LCDR1, 2 and 3 of the light chain variable domain set forth in SEQ ID NO:49. In another preferred embodiment, the antigen binding site that binds to CD3, e.g., a CD3 antigen binding site having the scFv or Fab structure described above, comprises the sequences of HCDR1, 2 and 3 of the heavy chain variable domain set forth in SEQ ID NO:50, and the sequences of LCDR1, 2 and 3 of the light chain variable domain set forth in SEQ ID NO:49.

[0090] In some preferred embodiments, an antigen binding site that binds to CD3, e.g., a CD3 antigen binding site having an scFv or Fab structure as described above, comprises the following: - HCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 41, - an HCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 42 or 47, - an HCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 43, - LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 44, - an LCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 45, - LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO: 46, Alternatively, said antigen binding site which binds to CD3 comprises a variant of one of said combinations of CDR sequences, wherein said variant comprises a total of at least one and not more than 5, 4, 3, 2 or 1 amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in 1, 2, 3, 4, 5 or preferably 6 CDR regions, preferably wherein the heavy chain CDR3 remains unchanged.

[0091] In some preferred embodiments, the antigen binding site that binds to CD3, e.g., a CD3 antigen binding site having an scFv or Fab structure as described above, comprises a heavy chain variable domain VH having the amino acid sequence set forth in SEQ ID NO: 48 or 50, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0092] In some preferred embodiments, the antigen binding site that binds to CD3, e.g., a CD3 antigen binding site having an scFv or Fab structure as described above, comprises a light chain variable domain VL having an amino acid sequence as set forth in SEQ ID NO:49 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto. In some preferred embodiments, the antigen binding site that binds to CD3, e.g. a CD3 antigen binding site having an scFv or Fab structure as described above, comprises a heavy chain variable domain VH having the amino acid sequence as set forth in SEQ ID NO: 48 or 50, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and comprises a light chain variable domain VL having the amino acid sequence as set forth in SEQ ID NO: 49, or an amino acid sequence with at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0093] Preferably, the antigen-binding site that binds to CD3, e.g., a CD3 antigen-binding site having an scFv or Fab structure as described above, comprises a VH domain having the amino acid sequence shown in SEQ ID NO: 48 or 50 and a VL domain having the amino acid sequence shown in SEQ ID NO: 49. More preferably, the antigen-binding site that binds to CD3, e.g., a CD3 antigen-binding site having an scFv or Fab structure as described above, comprises a VH domain having the amino acid sequence shown in SEQ ID NO: 48 and a VL domain having the amino acid sequence shown in SEQ ID NO: 49. In some preferred embodiments, when the antigen-binding site that binds to CD3 is a disulfide-stabilized scFv, said scFv further comprises a cysteine ​​substitution at position 44 in the VH domain and at position 100 in the VL domain.

[0094] The stem of the antibody molecule of the present invention The antibody molecule of the present invention comprises a stem located at the C-terminus of the antibody arm and formed by a first Fc domain and a second Fc domain. In some embodiments, the first and second Fc domains are the same. In other embodiments, the first and second Fc domains are different and are paired to form a heterodimer.

[0095] The Fc domain fragment of the antibody molecule applied to the present invention may be any antibody Fc domain. For example, the Fc domain of the antibody of the present invention may include two or three constant domains, namely, a CH2 domain, a CH3 domain and an optional CH4 domain. Preferably, the Fc domain of the antibody of the present invention includes CH2-CH3 from the N-terminus to the C-terminus, more preferably includes hinge region-CH2-CH3 from the N-terminus to the C-terminus. In the antibody molecules of the present invention, the Fc domain may comprise the second and third constant domains (CH2 and CH3 domains) derived from an IgG, IgA and IgD class antibody, or the second, third and fourth constant domains (CH2, CH3 and CH4 domains) derived from an IgM and IgE class antibody. In some embodiments, the Fc domain of the antibody molecule is an Fc domain from an IgG, such as an Fc domain from an IgG1, IgG2 or IgG4, preferably an Fc domain from human IgG1.

[0096] As will be appreciated by those skilled in the art, depending on the anticipated use of the antibody molecule of the present invention, the antibody molecule of the present invention may contain modifications in the Fc domain that alter effector functions. In one embodiment, one or more effector functions of the Fc domain of the present invention are reduced or eliminated compared to a wild-type Fc domain of the same isotype. The effector function of the Fc domain can be reduced or eliminated by any method selected from altering the glycosylation of the Fc domain, using an Fc isotype that naturally has reduced or eliminated effector functions, and modifying the amino acid sequence of the Fc domain.

[0097] In one embodiment, the glycosylation of the Fc domain is reduced to reduce or eliminate the effector function. Various methods for reducing the glycosylation of the Fc domain known in the art include, but are not limited to, producing the antibody molecule of the present invention in an environment that does not tolerate wild-type glycosylation, removing carbohydrate groups already present in the Fc domain, and modifying the Fc domain so that wild-type glycosylation does not occur. In one embodiment, the glycosylation of the Fc domain is reduced by modifying the Fc domain. For example, by introducing a mutation at position 297 of the Fc domain, the wild-type asparagine residue at that position is replaced with another amino acid that prevents glycosylation at that position, such as the N297A mutation.

[0098] In another embodiment, effector function is reduced or eliminated by modifying the amino acid sequence of at least one Fc domain. The Fc domain modifications introduce a point mutation at one or more positions selected from 238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 292, 293, 294, 295, 296, 297, 298, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, ​​388, 389, 414, 416, 419, 434, 435, 437, 438 and 439 that impairs binding of the Fc domain to one or more Fc receptors, or introduce a point mutation at a position selected from 270, 322, 329 and 321 that impairs C1q binding.

[0099] As will be appreciated by those skilled in the art, depending on the anticipated use of the antibody molecule of the invention, the antibody molecule of the invention may comprise modifications in the Fc domain that alter the binding affinity to one or more Fc receptors. In one embodiment, said Fc receptor is an Fcγ receptor, in particular a human Fcγ receptor. In one embodiment, said modifications reduce the effector function of the antibody molecule of the invention. In one specific embodiment, said effector function is antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the Fc domain of the antibody molecule of the invention comprises amino acid substitutions at positions 234 and 235 (EU numbering). In one specific embodiment, said amino acid substitutions are L234A and L235A (LALA mutation). In another embodiment, the Fc domain of the antibody molecule of the invention comprises an amino acid substitution at position 329 (EU numbering). In one specific embodiment, said amino acid substitution is P329G.

[0100] As will be understood by those skilled in the art, when the antibody molecule of the present invention is an asymmetric antibody molecule (e.g., an antibody molecule of Format_2 and Format_7), in order to promote correct formation of the asymmetric antibody molecule, the antibody molecule of the present invention may contain mutations in the first and second Fc domains that facilitate heterodimerization of the first and second Fc domains. For example, complementary knob and hole mutations can be introduced into the first and second Fc domains by the knob-in-hole technique.

[0101] Thus, in some embodiments, the invention provides trispecific antibody molecules, wherein the first Fc domain and the second Fc domain comprise first and second heterodimerization mutations, respectively, that promote pairing and heterodimerization of the first and second Fc domains. In some preferred embodiments, the first heterodimerization mutation of the first Fc domain comprises a Knob mutation and the second heterodimerization mutation of the second Fc domain comprises a Hole mutation that is complementary to said knob mutation, or the first heterodimerization mutation of the first Fc domain comprises a Hole mutation and the second heterodimerization mutation of the second Fc domain comprises a Knob mutation that is complementary to said hole mutation. In some preferred embodiments, the Knob mutation is T366W and the complementary hole mutation is T366S / L368A / Y407V.

[0102] In one embodiment, the present invention provides i) a homodimeric Fc-region of the human IgG1 subclass, optionally with the mutations L234A and L235A, or ii) a homodimeric Fc-region of the human IgG4 subclass, optionally with the mutations P329G, S228P and L235E, or iii) a heterodimeric Fc-region, a) one Fc-region polypeptide comprises the mutation T366W and another Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C; or c) a heterodimeric Fc-region, wherein one Fc-region polypeptide comprises the mutations T366W and S354C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C; or iv) a heterodimeric Fc-region of the human IgG1 subclass, in which both Fc-region polypeptides contain the mutations L234A and L235A; and a) one Fc-region polypeptide comprises the mutation T366W and another Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C; or c) a heterodimeric Fc-region of the human IgG1 subclass, wherein one Fc-region polypeptide comprises the mutations T366W and S354C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C; or v) a heterodimeric Fc-region of the human IgG4 subclass, in which both Fc-region polypeptides contain the mutations P329G, S228P and L235E; and a) one Fc-region polypeptide comprises the mutation T366W and another Fc-region polypeptide comprises the mutations T366S, L368A and Y407V, or b) one Fc-region polypeptide comprises the mutations T366W and Y349C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and S354C; or c) a heterodimeric Fc-region of the human IgG4 subclass, wherein one Fc-region polypeptide comprises the mutations T366W and S354C and another Fc-region polypeptide comprises the mutations T366S, L368A, Y407V and Y349C; The present invention provides a trispecific antibody molecule comprising:

[0103] In some embodiments, the Fc domain of the antibody molecule of the present invention may further comprise other mutations that facilitate purification of the heteropolymeric antibody, for example, an H435R mutation is introduced into one of the first and second Fc domains (e.g., the Fc domain with the Hole mutation) to facilitate purification of the target heteropolymeric antibody using Protein A.

[0104] In the antibody molecule of the present invention, the antibody stem is linked to the antibody arm at the N-terminus of the Fc domain. As will be apparent to those skilled in the art, in the antibody molecule applied to the present invention, the linking peptide linking the antibody arm and the stem Fc domain fragment may be any flexible linking peptide known in the art. In some embodiments, the linking peptide may comprise an amino acid sequence of a hinge region derived from IgG1, or may comprise an amino acid sequence selected from (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is an integer of at least 1. Preferably, the antibody stem may be linked to the antibody arm via a hinge region derived from IgG (particularly a hinge region derived from human IgG1). In some embodiments, for the heteropolymeric antibodies of the present invention that include a hinge region, a mutation, for example C220S, may be introduced into the hinge region to promote the formation of a target heteropolymeric antibody.

[0105] Exemplary Trispecific Antibody Molecules Format_2 In some embodiments, when p=0 and q=1, the antibody molecule of the invention has a structure of (M1:M2)-Fc::Fc-(X2)q. Thus, in said antibody molecule, there is only one conjugated moiety X2 conjugated to the stem. In said antibody molecule, preferably, antibody arms M1 and M2 comprise Fab antigen binding sites that bind to a first antigen and a second antigen, respectively, and conjugated moiety X2 comprises an scFv antigen binding site that binds to a third antigen, where the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0106] Thus, in some preferred embodiments, the present invention provides a trispecific Y-shaped antibody molecule, said antibody molecule comprising: (M1:M2)-Fc::Fc-(X2)q, (Format_2) where q=1, M1 and M2 represent the first and second antibody arms of an antibody molecule, respectively, and M1 and M2 comprise Fab or scFab, preferably Fab, that bind the first and second antigens, respectively; Fc::Fc represents the stem of an antibody molecule and is composed of a paired and dimerized first and second Fc domains, where the first and second antibody arms are linked to the N-terminus of the first and second Fc domains, respectively, either directly or via a linking peptide (preferably a hinge region); X2 represents a conjugated moiety to the C-terminus of the stem, wherein said conjugated moiety comprises an scFv that binds a third antigen, wherein X2 is conjugated to the C-terminus of a first Fc domain, either directly or via a linking peptide, or to the C-terminus of a second Fc domain; wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0107] In some embodiments, the invention provides trispecific antibody molecules having a Format_2 structure, wherein: (a): the antibody arm M1 and the antibody arm M2 of the molecule comprise a Fab that binds GPRC5D and a Fab that binds CD3, respectively, and the complex component X2 comprises an scFv that binds BCMA, and the molecule has the structure (Fab_GPRC5D:Fab_CD3)-Fc::Fc-(scFv_BCMA) (e.g. TS-F2-1, 3-6 molecules of the Examples), or (b): the antibody arms M1 and M2 of said molecule comprise a Fab that binds BCMA and a Fab that binds CD3, respectively, and the complex component X2 comprises an scFv that binds GPRC5D, said molecule having the structure (Fab_BCMA:Fab_CD3)-Fc::Fc-(scFv_GPRC5D) (e.g. the TS-F2-2 molecule of the Examples), or (c): The antibody arms M1 and M2 of the molecule comprise a Fab that binds GPRC5D and a Fab that binds BCMA, respectively, and the complex component X2 comprises an scFv that binds CD3, and the molecule has the structure (Fab_GPRC5D:Fab_BCMA)-Fc::Fc-(scFv_CD3).

[0108] In any embodiment of the Format_2 antibody molecule of the present invention described above, the Fab or scFv that binds to GPRC5D may be any suitable Fab or scFv that specifically binds to GPRC5D, for example a GPRC5D antigen binding site of the present invention having the scFv or Fab structure described above, in particular an scFv or Fab having the VH and VL amino acid sequences described above.

[0109] In any embodiment of the Format_2 antibody molecule of the invention as described above, the Fab or scFv that binds BCMA may be any suitable Fab or scFv that specifically binds BCMA, for example a BCMA antigen binding site of the invention having an scFv or Fab structure as described above, in particular an scFv or Fab having the VH and VL amino acid sequences as described above.

[0110] In any of the embodiments of the Format_2 antibody molecule of the invention described above, the antigen binding site that binds to CD3 may be any suitable scFv or Fab that binds to a specific CD3, such as a CD3 antigen binding site of the invention having an scFv or Fab structure as described above, in particular an scFv or Fab having the VH and VL amino acid sequences as described above.

[0111] In any embodiment of the Format_2 antibody molecule of the present invention described above, the antibody molecule may comprise any of the antibody stem structures of the present invention that apply to Format_2 described above. For example, in the Format_2 asymmetric trispecific antibody molecule of the present invention, preferably, in order to promote correct pairing of the antibody molecule polypeptide chains, the first and second Fc domains of the stem may be introduced with mutations that can promote heterodimerization of the first and second Fc domains, in particular complementary "knobs-in-hole" mutations. For example, a knob mutation may be introduced in the first Fc domain and a complementary hole mutation may be introduced in the second Fc domain, or vice versa. In this way, the two Fc domains of the antibody molecule may pair to form a stable "knobs-in-hole" bond. Depending on the anticipated use, the first and / or second Fc domains of the Format_2 antibody molecule of the present invention may preferably further comprise mutations that affect antibody effector functions, such as mutations that reduce ADCC activity, for example LALA mutations.

[0112] In some embodiments, the Format_2 trispecific antibody molecule of the invention comprises or consists of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; the first light chain polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable domain, VL, an immunoglobulin CL domain; the second heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; the second light chain polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable domain VL, an immunoglobulin CL domain; and wherein the first heavy chain polypeptide chain or the second heavy chain polypeptide chain further comprises an scFv domain conjugated to the C-terminus of the Fc domain, either directly or preferably via a linking peptide (e.g., (G4S)2 or TS(G4S)2), Where: the VH-CH1 of the first heavy polypeptide chain and the VL-CL of the first light polypeptide chain pair to form a first antibody arm (M1) that binds to a first antigen; the VH-CH1 of the second heavy polypeptide chain and the VL-CL of the second light polypeptide chain pair to form a second antibody arm (M2) that binds to a second antigen; the Fc domain of the first heavy polypeptide chain and the Fc domain of the second heavy polypeptide chain pair and dimerize to form an antibody stem (Fc::Fc); and the scFv domain conjugated to the C-terminus of the first or second heavy chain polypeptide chain forms a conjugate (X2) that binds to a third antigen; wherein the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0113] In one embodiment, the antibody arm M1 binds to GPCR5D, the second antibody arm M2 binds to CD3, and the conjugate moiety X2 binds to BCMA, thus the first heavy polypeptide chain and the first light polypeptide chain each contain, at their N-terminus, a VH and a VL amino acid sequence of a Fab antigen-binding site that specifically binds to GPRC5D; the second heavy chain and the second light chain each contain at their N-terminus the VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to CD3; Wherein, when conjugate moiety X2 is conjugated to a first heavy chain polypeptide chain, the first heavy chain polypeptide chain further comprises, at its C-terminus, the amino acid sequence of an scFv that specifically binds to BCMA, or, preferably, when conjugate moiety X2 is conjugated to a second heavy chain polypeptide chain, the second heavy chain polypeptide chain further comprises, at its C-terminus, the amino acid sequence of an scFv that specifically binds to BCMA.

[0114] In another embodiment, the antibody arm M1 binds to BCMA, the second antibody arm M2 binds to CD3, and the conjugate moiety X2 binds to GPCR5D, thus the first heavy polypeptide chain and the first light polypeptide chain each contain at their N-terminus VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to BCMA; the second heavy chain and the second light chain each contain at their N-terminus the VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to CD3; Here, when conjugate component X2 is conjugated to a first heavy chain polypeptide chain, the first heavy chain polypeptide chain further comprises, at its C-terminus, the amino acid sequence of an scFv that specifically binds to GPRC5D; or, preferably, when conjugate component X2 is conjugated to a second heavy chain polypeptide chain, the second heavy chain polypeptide chain further comprises, at its C-terminus, the amino acid sequence of an scFv that specifically binds to GPRC5D.

[0115] In a further embodiment, the antibody arm M1 is bound to GPCR5D, the second antibody arm M2 is bound to BCMA and the conjugate moiety X2 is bound to CD3, thus the first heavy polypeptide chain and the first light polypeptide chain each contain, at their N-terminus, a VH and a VL amino acid sequence of a Fab antigen-binding site that specifically binds to GPRC5D; the second heavy chain polypeptide chain and the second light chain polypeptide chain each contain at their N-terminus the VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to BCMA; Here, when conjugate component X2 is conjugated to a first heavy chain polypeptide chain, the first heavy chain polypeptide chain further comprises, at its C-terminus, the amino acid sequence of an scFv that specifically binds to CD3; or, when conjugate component X2 is conjugated to a second heavy chain polypeptide chain, the second heavy chain polypeptide chain further comprises, at its C-terminus, the amino acid sequence of an scFv that specifically binds to CD3.

[0116] In a preferred embodiment, the Fab or scFv that specifically binds to GPRC5D is selected from the following group: (a) VH comprising HCDR1 to HCDR3 sequences of SEQ ID NOs: 1 to 3 and VL comprising LCDR1 to HCDR3 sequences of SEQ ID NOs: 4 to 6, (b) VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 7 to 8 and VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 10 to 12; (c) a VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 13 to 15 and a VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 16 to 18, or (d) VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 19 to 21 and VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 22 to 24; and VH and VL selected from Preferably, said Fab and said scFv comprise a VH and VL amino acid sequence pair selected from the following group: SEQ ID NO: 25 / 26, SEQ ID NO: 27 / 28, SEQ ID NO: 29 / 30 and SEQ ID NO: 31 / 32, And preferably, when the scFv is a dsscFv, it further comprises cysteine ​​substitutions introduced into said VH and VL sequences, for example a cysteine ​​substitution at position 44 of the VH sequence and at position 100 of the VL sequence. In a preferred embodiment, the Fab or scFv that specifically binds to CD3 is selected from the following group: (a) a VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 41 to 43 and a VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 44 to 46, or (b) VH comprising HCDR1 to HCDR3 sequences of SEQ ID NOs: 41, 47, and 43, and VL comprising LCDR1 to HCDR3 sequences of SEQ ID NOs: 44 to 46; and VH and VL selected from Preferably, said Fab and scFv comprise a VH and VL amino acid sequence pair selected from the following group: SEQ ID NO: 48 / 49, SEQ ID NO: 50 / 49, and more preferably SEQ ID NO: 48 / 49; And preferably, when the scFv is a dsscFv, it further comprises cysteine ​​substitutions introduced into said VH and VL sequences, for example a cysteine ​​substitution at position 44 of the VH sequence and at position 100 of the VL sequence.

[0117] In a preferred embodiment, the Fab or scFv that specifically binds to BCMA comprises: VH comprising HCDR1 to HCDR3 sequences of SEQ ID NOs: 33 to 35 and VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 36 to 38, Preferably, the Fab and scFv comprise the VH and VL amino acid sequence pair SEQ ID NO: 39 / 40, And preferably, when the scFv is a dsscFv, it further comprises cysteine ​​substitutions introduced into said VH and VL sequences, for example a cysteine ​​substitution at position 44 of the VH sequence and at position 100 of the VL sequence.

[0118] In some preferred embodiments, the first light chain polypeptide chain and the second light chain polypeptide chain comprise a kappa light chain constant domain, CL κ and the lamda light chain constant domain CL λ In one specific embodiment, the light chain constant domain CL κ comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto, and a light chain constant domain CL λ comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0119] In some preferred embodiments, the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise a heavy chain constant region CH1 derived from human IgG1, and preferably comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0120] In some preferred embodiments, the first heavy chain polypeptide chain and the second heavy chain polypeptide chain each comprise an Fc domain comprising a knob or a complementary hole mutation. In a specific embodiment, the Fc domain comprising a knob mutation comprises T366W, and the Fc domain comprising a complementary hole mutation comprises T366S, L368A, and Y407V mutations. In a specific embodiment, the Fc domain comprising a hole mutation comprises an amino acid sequence selected from SEQ ID NOs: 102 and 107, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto, and the Fc domain comprising a complementary knob mutation comprises an amino acid sequence of SEQ ID NO: 103, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0121] In some embodiments, the present invention provides a trispecific antibody molecule of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:65, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:66, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:67, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0122] In some embodiments, the present invention provides a trispecific antibody molecule of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:69, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0123] In some embodiments, the present invention provides a trispecific antibody molecule of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 74, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0124] In some embodiments, the present invention provides a trispecific antibody molecule of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77 or 78, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0125] In some embodiments, the present invention provides a trispecific antibody molecule of Format_2 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:79, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0126] Format_7 In some embodiments, when q=0 and p=2, the antibody molecule of the invention has the structure (M1:M2-(X1)p)-Fc::Fc. Thus, in said antibody molecule, there is only a conjugate moiety X1 conjugated to an antibody arm, in which antibody arms M1 and M2 comprise Fab antigen binding sites that bind a first antigen and a second antigen, respectively, and conjugate moiety X1 comprises an scFv antigen binding site that binds a third antigen, where the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0127] Thus, in some preferred embodiments, the present invention provides a trispecific Y-shaped antibody molecule, said antibody molecule comprising: (M1:M2-(X1)p)-Fc::Fc, (Format_7) wherein p=2; M1 and M2 represent a first antibody arm and a second antibody arm, respectively, of an antibody molecule, and M1 and M2 comprise Fabs that bind to a first and a second antigen, respectively; Fc::Fc represents the stem of an antibody molecule and is composed of a paired and dimerized first and second Fc domains, where the first and second antibody arms are linked to the N-terminus of the first and second Fc domains, respectively, either directly or via a linking peptide (preferably a hinge region); X1 represents a conjugated moiety to the C-terminus of an antibody arm, said conjugated moiety comprising an scFv that binds to a third antigen, wherein X1 is conjugated directly or via a linking peptide to the C-terminus of the Fab light chain of the first and second antibody arms; wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0128] In some embodiments, the invention provides trispecific antibody molecules having a Format_7 structure, wherein: (a): the antibody arm M1 and the antibody arm M2 of the molecule comprise a Fab that binds GPRC5D and a Fab that binds CD3, respectively, and the complex component X1 comprises an scFv that binds BCMA, and the molecule has the structure (Fab_GPRC5D:Fab_CD3-(scFv_BCMA)2)-Fc::Fc (e.g. the molecules of examples TS-F7-1, 2, 5), or (b): the antibody arm M1 and the antibody arm M2 of the molecule comprise a Fab that binds GPRC5D and a Fab that binds BCMA, respectively, and the complex component X1 comprises an scFv that binds CD3, and the molecule has the structure (Fab_GPRC5D:Fab_BCMA-(scFv_CD3)2)-Fc::Fc (e.g., the molecules of examples TS-F7-3,4), or (c) antibody arm M1 and antibody arm M2 of the molecule comprise a Fab that binds BCMA and a Fab that binds CD3, respectively, and complex moiety X1 comprises an scFv that binds GPRC5D, and the molecule has the structure (Fab_BCMA:Fab_CD3-(scFv_GPRC5D)2)-Fc::Fc.

[0129] In any embodiment of the Format_7 antibody molecule of the present invention described above, the Fab or scFv that binds to GPRC5D may be any suitable Fab or scFv that specifically binds to GPRC5D, for example a GPRC5D antigen binding site of the present invention having the scFv or Fab structure described above, in particular an scFv or Fab having the VH and VL amino acid sequences described above. In any embodiment of the Format_7 antibody molecule of the invention as described above, the Fab or scFv that binds BCMA may be any suitable Fab or scFv that specifically binds BCMA, such as a BCMA antigen binding site of the invention having the scFv or Fab structure as described above, in particular an scFv or Fab having the VH and VL amino acid sequences as described above. In any of the embodiments of the Format_7 antibody molecule of the invention described above, the antigen binding site that binds to CD3 may be any suitable scFv or Fab that specifically binds to CD3, such as a CD3 antigen binding site of the invention having the scFv or Fab structure described above, in particular an scFv or Fab having the VH and VL amino acid sequences described above.

[0130] In any embodiment of the Format_7 antibody molecule of the invention described above, the antibody molecule may comprise any of the antibody stem structures of the invention that apply to Format_7 described above. For example, in the Format_7 asymmetric trispecific antibody molecule of the invention, preferably, the first and second Fc domains of the stem may be introduced with mutations that can promote heterodimerization of the first and second Fc domains, in particular complementary "knobs-in-hole" mutations, to promote correct pairing of the antibody molecule polypeptide chains. For example, a knob mutation may be introduced in the first Fc domain and a complementary hole mutation may be introduced in the second Fc domain, or vice versa. In this way, the two Fc domains of the antibody molecule may pair to form a stable "knobs-in-hole" bond. Depending on the anticipated use, the first and / or second Fc domains of the Format_7 antibody molecule of the invention may preferably further comprise mutations that affect antibody effector functions, such as mutations that reduce ADCC activity, for example LALA mutations.

[0131] In some embodiments, the Format_7 trispecific antibody molecule of the invention comprises or consists of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; the first light chain polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable domain, VL, an immunoglobulin CL domain; the second heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; the second light chain polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable domain VL, an immunoglobulin CL domain; and wherein the first light chain polypeptide chain and the second light chain polypeptide chain further comprise an scFv domain linked to the C-terminus of the CL domain thereof, either directly or preferably via a linking peptide (e.g., (G4S)2 or TS(G4S)2); Where: the VH-CH1 of the first heavy polypeptide chain and the VL-CL of the first light polypeptide chain pair to form a first antibody arm (M1) that binds to a first antigen; the VH-CH1 of the second heavy polypeptide chain and the VL-CL of the second light polypeptide chain pair to form a second antibody arm (M1) that binds to a second antigen; the Fc domain of the first heavy polypeptide chain and the Fc domain of the second heavy polypeptide chain pair and dimerize to form an antibody stem (Fc::Fc); and the scFv domain conjugated to the C-terminus of the first light polypeptide chain and the second light polypeptide chain forms a conjugate (X1) that binds to a third antigen; wherein the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0132] In one embodiment, the antibody arm M1 binds to GPCR5D, the second antibody arm M2 binds to CD3, and the conjugate moiety X1 binds to BCMA, thus the first heavy polypeptide chain and the first light polypeptide chain each contain, at their N-terminus, a VH and a VL amino acid sequence of a Fab antigen-binding site that specifically binds to GPRC5D; the second heavy chain and the second light chain each contain at their N-terminus the VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to CD3; Furthermore, the first light chain polypeptide chain and the second light chain polypeptide chain further comprise, at their C-terminus, an amino acid sequence of an scFv that specifically binds to BCMA.

[0133] In one embodiment, the antibody arm M1 binds to BCMA, the second antibody arm M2 binds to CD3, and the conjugate moiety X1 binds to GPCR5D, thus the first heavy polypeptide chain and the first light polypeptide chain each contain at their N-terminus VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to BCMA; the second heavy chain and the second light chain each contain at their N-terminus the VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to CD3; Furthermore, the first light chain polypeptide chain and the second light chain polypeptide chain further comprise, at their C-terminus, an amino acid sequence of an scFv that specifically binds to GPRC5D.

[0134] In one embodiment, the antibody arm M1 binds to GPCR5D, the second antibody arm M2 binds to BCMA, and the conjugate moiety X1 binds to CD3, thus the first heavy polypeptide chain and the first light polypeptide chain each contain, at their N-terminus, a VH and a VL amino acid sequence of a Fab antigen-binding site that specifically binds to GPRC5D; the second heavy chain polypeptide chain and the second light chain polypeptide chain each contain at their N-terminus the VH and VL amino acid sequences of a Fab antigen-binding site that specifically binds to BCMA; Furthermore, the first light chain polypeptide chain and the second light chain polypeptide chain further comprise, at their C-terminus, an amino acid sequence of an scFv that specifically binds to CD3.

[0135] In a preferred embodiment, the Fab or scFv that specifically binds to GPRC5D is selected from the following group: (a) VH comprising HCDR1 to HCDR3 sequences of SEQ ID NOs: 1 to 3 and VL comprising LCDR1 to HCDR3 sequences of SEQ ID NOs: 4 to 6, (b) VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 7 to 8 and VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 10 to 12; (c) a VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 13 to 15 and a VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 16 to 18, or (d) VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 19 to 21 and VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 22 to 24; and VH and VL selected from Preferably, said Fab and said scFv comprise a VH and VL amino acid sequence pair selected from the following group: SEQ ID NO: 25 / 26, SEQ ID NO: 27 / 28, SEQ ID NO: 29 / 30 and SEQ ID NO: 31 / 32, And preferably, when the scFv is a dsscFv, it further comprises cysteine ​​substitutions introduced into said VH and VL sequences, for example a cysteine ​​substitution at position 44 of the VH sequence and at position 100 of the VL sequence.

[0136] In a preferred embodiment, the Fab or scFv that specifically binds to CD3 is selected from the following group: (a) a VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 41 to 43 and a VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 44 to 46, or (b) VH comprising HCDR1 to HCDR3 sequences of SEQ ID NOs: 41, 47, and 43, and VL comprising LCDR1 to HCDR3 sequences of SEQ ID NOs: 44 to 46; and VH and VL selected from Preferably, said Fab and scFv comprise a VH and VL amino acid sequence pair selected from the following group: SEQ ID NO: 48 / 49, SEQ ID NO: 50 / 49, and more preferably SEQ ID NO: 48 / 49; And preferably, when the scFv is a dsscFv, it further comprises cysteine ​​substitutions introduced into said VH and VL sequences, for example a cysteine ​​substitution at position 44 of the VH sequence and at position 100 of the VL sequence.

[0137] In a preferred embodiment, the Fab or scFv that specifically binds to BCMA comprises: VH comprising HCDR1 to HCDR3 sequences of SEQ ID NOs: 33 to 35 and VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 36 to 38, Preferably, the Fab and scFv comprise the VH and VL amino acid sequence pair SEQ ID NO: 39 / 40, And preferably, when the scFv is a dsscFv, it further comprises cysteine ​​substitutions introduced into said VH and VL sequences, for example a cysteine ​​substitution at position 44 of the VH sequence and at position 100 of the VL sequence.

[0138] In some embodiments, the first light chain polypeptide chain and the second light chain polypeptide chain comprise a kappa light chain constant domain, CL κ In some embodiments, the first light chain polypeptide chain and the second light chain polypeptide chain comprise a lamda light chain constant domain, CL λ In one specific embodiment, the light chain constant domain CL κ In one specific embodiment, the light chain constant domain CL comprises the amino acid sequence of SEQ ID NO: 105 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto. λ comprises the amino acid sequence of SEQ ID NO: 106 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0139] In some preferred embodiments, the first heavy chain polypeptide chain and the second heavy chain polypeptide chain comprise a heavy chain constant region CH1 derived from human IgG1, and preferably comprises the amino acid sequence of SEQ ID NO: 104, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0140] In some preferred embodiments, the first heavy chain polypeptide chain and the second heavy chain polypeptide chain each comprise an Fc domain comprising a knob or a complementary hole mutation. In a specific embodiment, the Fc domain comprising a knob mutation comprises T366W, and the Fc domain comprising a complementary hole mutation comprises T366S, L368A, and Y407V mutations. In a specific embodiment, the Fc domain comprising a knob mutation comprises an amino acid sequence selected from SEQ ID NOs: 102 and 107, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto, and the Fc domain comprising a complementary hole mutation comprises an amino acid sequence of SEQ ID NO: 103, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0141] In some embodiments, the present invention provides a trispecific antibody molecule of Format_7 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:87, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77 or 78, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:89, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0142] In some embodiments, the present invention provides a trispecific antibody molecule of Format_7 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:91, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:89, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0143] In some embodiments, the present invention provides a trispecific antibody molecule of Format_7 structure comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:92, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 93 or 96, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:94, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second light chain polypeptide chain comprises the amino acid sequence of SEQ ID NO: 95 or 97, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0144] Format_1 In some embodiments, when p=0 and q=1, the antibody molecule of the invention has the structure (M1:M2)-Fc::Fc-(X2)q, where antibody arms M1, M2 and composite moiety X2 comprise scFv antigen binding sites that bind to a first antigen, a second antigen and a third antigen, respectively, where the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0145] Thus, in some preferred embodiments, the present invention provides a trispecific Y-shaped antibody molecule, said antibody molecule comprising: (M1:M2)-Fc::Fc-(X2)q, (Format_1) wherein q=1; M1 and M2 represent a first antibody arm and a second antibody arm of an antibody molecule, respectively, and M1 and M2 comprise scFvs that bind to a first and a second antigen, respectively; Fc::Fc represents the stem of an antibody molecule and is composed of a paired and dimerized first and second Fc domains, where the first and second antibody arms are linked to the N-terminus of the first and second Fc domains, respectively, either directly or via a linking peptide (preferably a hinge region); X2 represents a conjugated moiety to the C-terminus of the stem, wherein said conjugated moiety comprises an scFv that binds a third antigen, wherein X2 is conjugated to the C-terminus of a first Fc domain, either directly or via a linking peptide, or to the C-terminus of a second Fc domain; wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0146] In some embodiments, the invention provides trispecific antibody molecules having a Format_1 structure, wherein: (a): the antibody arm M1 and the antibody arm M2 of the molecule comprise an scFv that binds GPRC5D and an scFv that binds CD3, respectively, and the complex component X2 comprises an scFv that binds BCMA, and the molecule has the structure (scFv_GPRC5D:scFv_CD3)-Fc::Fc-(scFv_BCMA) (e.g. the molecule of example TS-F1-1), or (b): antibody arm M1 and antibody arm M2 of the molecule comprise an scFv that binds BCMA and an scFv that binds CD3, respectively, and complex component X2 comprises an scFv that binds GPRC5D, and the molecule has the structure (scFv_BCMA:scFv_CD3)-Fc::Fc-(scFv_GPRC5D) (e.g., the molecule of example TS-F1-2).

[0147] In some embodiments, the Format_1 trispecific antibody molecule of the invention comprises or consists of a first heavy chain polypeptide chain and a second heavy chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a first scFv and an Fc domain; the second heavy chain polypeptide chain comprises, from the N-terminus to the C-terminus, a second scFv and an Fc domain; and wherein the first heavy chain polypeptide chain or the second heavy chain polypeptide chain further comprises a third scFv domain conjugated to the C-terminus of the Fc domain, either directly or preferably via a linking peptide (e.g., (G4S)2 or TS(G4S)2); Where: a first scFv of the first heavy polypeptide chain forms a first antibody arm (M1) that binds to a first antigen; a second scFv of the second heavy chain polypeptide chain forms a second antibody arm (M1) that binds to a second antigen; the Fc domain of the first heavy polypeptide chain and the Fc domain of the second heavy polypeptide chain pair and dimerize to form an antibody stem (Fc::Fc); and a third scFv domain conjugated to the C-terminus of the first heavy chain polypeptide chain or the second heavy chain polypeptide chain forms a conjugate (X2) that binds to a third antigen; wherein the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0148] In some embodiments, the present invention provides trispecific antibody molecules having a Format_1 structure comprising, or consisting of, a first heavy chain polypeptide chain and a second heavy chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:61, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:62, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0149] In some embodiments, the present invention provides trispecific antibody molecules having a Format_1 structure comprising, or consisting of, a first heavy chain polypeptide chain and a second heavy chain polypeptide chain, wherein: the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:63, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The second heavy chain polypeptide chain comprises the amino acid sequence of SEQ ID NO:64, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0150] Format_6 In some embodiments, when p=2 and q=2, the antibody molecule of the invention has the structure (M1:M2-(X2)p)-Fc::Fc-(X2)q, where antibody arms M1 and M2 comprise a Fab antigen binding site that binds a first antigen, and composite moieties X1 and X2 comprise scFv antigen binding sites that bind a second and third antigen, respectively, where the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0151] Thus, in some preferred embodiments, the present invention provides a trispecific symmetric Y-shaped antibody molecule, said antibody molecule comprising: (M1:M2-(X2)p)-Fc::Fc-(X2)q, (Format_6) wherein p=2 and q=2; M1 and M2 represent a first antibody arm and a second antibody arm, respectively, of an antibody molecule, and both M1 and M2 comprise a Fab that binds to a first antigen; Fc::Fc represents the stem of an antibody molecule and is composed of a paired and dimerized first and second Fc domains, where the first and second antibody arms are linked to the N-terminus of the first and second Fc domains, respectively, either directly or via a linking peptide (preferably a hinge region); X1 and X2 represent conjugated moieties to the C-terminus of an antibody arm Fab light chain or the C-terminus of a stem Fc domain, respectively, where said conjugation moiety X1 comprises an scFv that binds a second antigen and said conjugation moiety X2 comprises an scFv that binds a third antigen, where X1 and X2 are conjugated to the arm or stem directly or via a linking peptide; wherein the first, second and third antigens are different from each other and independently selected from GPRC5D, BCMA and CD3.

[0152] In some embodiments, the invention provides trispecific antibody molecules having a Format_6 structure, wherein: (a) the antibody arm M1 and the antibody arm M2 of the molecule comprise a Fab that binds to GPRC5D, the complex component X1 comprises an scFv for CD3, and the complex component X2 comprises an scFv that binds to BCMA, the molecule having the structure (Fab_GPRC5D:Fab_GPRC5D-(scFv_CD3)2)-Fc::Fc-(scFv_BCMA)2 (e.g. the molecules of examples TS-F6-1,2), or (b): antibody arm M1 and antibody arm M2 of the molecule comprise a Fab that binds BCMA, complex component X1 comprises an scFv of CD3 and complex component X2 comprises an scFv that binds GPRC5D, the molecule having the structure (Fab_BCMA:Fab_BCMA-(scFv_CD3)2)-Fc::Fc-(scFv_GPRC5D)2 (e.g. the molecules of examples TS-F6-3,4).

[0153] In some embodiments, the Format_6 trispecific antibody molecule of the invention comprises or consists of two identical heavy polypeptide chains and two identical light polypeptide chains, wherein: the heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain, an Fc domain, and a first scFv domain; the light chain polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable domain VL, an immunoglobulin CL domain, and a second scFv domain; wherein the first and second scFv domains of the heavy and light polypeptide chains are conjugated to their C-terminus, respectively, either directly or preferably via a linking peptide (e.g., (G4S)2 or TS(G4S)2); Where: a VH-CH1 domain at the N-terminus of one heavy polypeptide chain and a VL-CH1 domain at the N-terminus of one light polypeptide chain pair to form a first antibody arm (M1) that binds to a first antigen; the N-terminal VH-CH1 domain of another heavy chain polypeptide chain and the N-terminal VL-CH1 domain of one light chain polypeptide chain pair to form a second antibody arm (M2) that binds to the first antigen; The Fc domains of the two heavy polypeptide chains pair and dimerize to form the antibody stem (Fc::Fc); and The scFv domains conjugated to the C-terminus of the two light polypeptide chains form a conjugate (X1) that binds to a second antigen, The scFv domains attached to the C-terminus of the two heavy polypeptide chains form a complex (X2) that binds to a third antigen, wherein the first, second and third antigens are each different and independently selected from GPRC5D, BCMA and CD3.

[0154] In some embodiments, the present invention provides a trispecific antibody molecule of Format_6 structure comprising or consisting of two identical heavy polypeptide chains and two identical light polypeptide chains, wherein: the heavy polypeptide chain comprises an amino acid sequence of SEQ ID NO: 81, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The light polypeptide chain comprises an amino acid sequence of SEQ ID NO: 82 or 83, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0155] In some embodiments, the present invention provides a trispecific antibody molecule of Format_6 structure comprising or consisting of two identical heavy polypeptide chains and two identical light polypeptide chains, wherein: the heavy polypeptide chain comprises an amino acid sequence of SEQ ID NO: 84, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The light polypeptide chain comprises an amino acid sequence of SEQ ID NO: 85 or 86, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto.

[0156] III. GPRC5D Antibody Molecules of the Invention In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to GPRC5D. In some preferred embodiments, the GPRC5D antibody or antigen-binding fragment thereof of the present invention is (i) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 25 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 26, or (ii) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 27 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 28, or (iii) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 29 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 30; or (iv) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 31 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 32; or (v) the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 98 and LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 99; or (vi) comprises the sequences of HCDR1, 2 and 3 of the heavy chain variable domain as set forth in SEQ ID NO: 100, and the sequences of LCDR1, 2 and 3 of the light chain variable domain as set forth in SEQ ID NO: 101.

[0157] In some other embodiments, the GPRC5D antibody or antigen-binding fragment thereof of the invention comprises: (i) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 1 to 6, respectively; or (ii) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 7 to 12, respectively; or (iii) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 13 to 18, respectively; or (iv) the sequences of heavy chain variable domains HCDR1, 2 and 3 and light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 19 to 24, respectively; or (v) comprising a combination of CDR sequences selected from the sequences of heavy chain variable domains HCDR1, 2 and 3 and the sequences of light chain variable domains LCDR1, 2 and 3 shown in SEQ ID NOs: 13, 110 and 18, respectively.

[0158] In some further embodiments, the GPRC5D antibody molecule or antigen-binding fragment thereof of the present invention comprises: (a) a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO:25, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO:26, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (b) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (c) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (d) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (e) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 98, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 99, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (f) a combination of VH and VL amino acid sequences selected from: a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 100, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 101, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto.

[0159] Preferably, the GPRC5D antibody molecule or antigen-binding fragment thereof of the present invention comprises: (i) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 25, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 26, or (ii) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 27, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 28, or (iii) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 29, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 30; or (iv) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 32; (v) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 98, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 99; (vi) a combination of VH and VL amino acid sequences selected from a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 100, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 101.

[0160] In some embodiments, the GPRC5D antibody of the present invention is a monospecific antibody, a bispecific antibody, or a trispecific antibody. In some preferred embodiments, the GPRC5D antibody of the present invention is a bispecific antibody that further comprises an antigen binding site that binds to CD3, such as the CD3 antigen binding site of the present invention described above. In another preferred embodiment, the GPRC5D antibody of the present invention is a trispecific antibody that further comprises an antigen binding site that binds to BCMA (such as the BCMA antigen binding site of the present invention described above) and an antigen binding site that binds to CD3 (such as the CD3 antigen binding site of the present invention described above).

[0161] In some embodiments, the GPRC5D antibody of the invention is a bispecific antibody that binds to GPRC5D and CD3, Preferably, wherein the antibody is selected from the following group that binds to CD3: (a) a VH comprising HCDR1 to 3 sequences of SEQ ID NOs: 41 to 43 and a VL comprising LCDR1 to 3 sequences of SEQ ID NOs: 44 to 46, or (b) VH comprising HCDR1 to HCDR3 sequences of SEQ ID NOs: 47, 42, and 43, and VL comprising LCDR1 to HCDR3 sequences of SEQ ID NOs: 44 to 46; comprising a VH and VL amino acid sequence pair selected from Preferably, the VH and VL amino acid sequence pairs are selected from the following group: SEQ ID NO: 48 / 49 and SEQ ID NO: 50 / 49, Optionally, the VH and VL sequences include an introduced cysteine ​​substitution, for example, at position 44 of the VH sequence and at position 100 of the VL sequence, thus forming a disulfide bond between the VH and VL.

[0162] Thus, in a preferred embodiment, the present invention provides a bispecific antibody that binds to GPRC5D and CD3, wherein the antibody comprises a combination of a VH and VL amino acid sequence pair that binds to GPRC5D and a VH and VL amino acid sequence pair that binds to CD3 selected from the following group: [Table 2]

[0163] The invention further provides variants of the bispecific antibodies, wherein the variants comprise amino acid mutations, such as amino acid substitutions, deletions and / or insertions, in the VH and VL amino acid sequence pair that binds to GPRC5D and / or the VH and VL amino acid sequence pair that binds to CD3, wherein the amino acid mutations do not affect the binding of the bispecific antibody to GPRC5D and CD3. In some embodiments, the variants comprise a combination of amino acid sequences selected from the following: [Table 3-1] [Table 3-2]

[0164] In a preferred embodiment, the bispecific antibody of the present invention comprises a cysteine ​​substitution introduced into the VH and VL amino acid sequence pair that binds to GPRC5D and / or the VH and VL amino acid sequence pair that binds to CD3, for example a cysteine ​​substitution at position 44 of the VH sequence and at position 100 of the VL sequence, thus forming a disulfide bond between the VH and VL.

[0165] The present invention further provides fusion proteins and immunoconjugates (e.g., conjugates with toxins or small chemical molecules) comprising the GPRC5D antibodies of the present invention, as well as pharmaceutical compositions and drug combinations. In the pharmaceutical compositions or drug combinations, the antibodies of the present invention may include other therapeutic agents, such as other therapeutic agents applicable to the anticipated uses of the antibodies of the present invention, e.g., chemotherapeutic agents, radiotherapeutic agents, tumor suppressor molecules.

[0166] IV. Production and purification of antibody molecules of the invention In a further aspect, the invention provides a method for producing an antibody molecule of the invention, said method comprising culturing a host cell comprising a polynucleotide encoding a polypeptide chain under suitable polypeptide chain conditions to express said antibody, and assembling said polypeptide chains under suitable conditions for assembly of said polypeptide chains into said antibody molecule to produce said antibody.

[0167] When the antibody molecule of the present invention is an asymmetric antibody molecule, such as a trispecific antibody molecule comprising a first heavy chain polypeptide chain and a first light chain polypeptide chain, and a second heavy chain polypeptide chain and a second light chain polypeptide chain, e.g., an antibody molecule of Format_2 or Format_7, preferably, a method for producing the antibody molecule of the present invention comprises the steps of (i) culturing a host cell comprising one encoding the first heavy chain and one encoding the first light chain under conditions of a first heavy chain polypeptide chain and a first light chain polypeptide chain suitable for expressing the antibody molecule to produce a first parent protein, (ii) culturing a host cell comprising one encoding the second heavy chain and one encoding the second light chain under conditions of a second heavy chain polypeptide chain and a second light chain polypeptide chain suitable for expressing the antibody molecule to produce a second parent protein, and (iii) mixing the first parent protein and the second parent protein in an equimolar ratio and assembling them in vitro under suitable redox conditions to form the antibody. In one specific embodiment, step (iii) includes adding glutathione (GSH) to the mixed solution of the purified first and second parent proteins, preferably controlling the molar ratio of GSH / protein to 500-700. In one specific embodiment, step (iii) further includes removing GSH followed by natural oxidation for a certain period of time (e.g., 2-3 hours).

[0168] The polypeptide chains of the antibody molecules of the invention can be produced, for example, by solid phase peptide synthesis (e.g., Merrifield solid phase synthesis) or recombinantly. In recombinant production, polynucleotides encoding any one and / or more polypeptide chains of the antibody molecule are isolated and inserted into one or more vectors for cloning and / or expression in a host cell. The polynucleotides can be conveniently isolated and sequenced using routine methods. In one embodiment, there is provided a vector, preferably an expression vector, comprising one or more polynucleotides of the invention.

[0169] Expression vectors can be constructed using methods known to those skilled in the art, including, but not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). Once an expression vector containing one or more polynucleotides of the invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this goal, including, for example, protoplast fusion, calcium phosphate co-precipitation, electroporation, retroviral transduction, viral transfection, particle gun, liposome-based transfection, or other common techniques.

[0170] In one embodiment, one or more host cells are provided that comprise a polynucleotide of the invention. In some embodiments, a host cell is provided that comprises an expression vector of the invention. The term "host cell" as used herein refers to any type of cell line that can be engineered to produce an antibody molecule of the invention. Suitable host cells that support the replication and expression of the antibody molecule of the invention are known in the art. If necessary, such cells can be transfected or transduced with a particular expression vector, and many cells containing the vector can be cultured to inoculate large fermenters to obtain sufficient quantities of the antibody molecule of the invention for clinical use. Suitable host cells include prokaryotic microorganisms such as E. coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of mammalian host cell lines that can be used include SV40 transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (HEK 293 or 293F cells), baby hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NS0, P3X63 and Sp2 / 0, etc. For an overview of mammalian host cell lines suitable for protein production, see, for example, Yazaki & Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ), p. 255-268 (2003). In a preferred embodiment, the host cell is a CHO, HEK293 or NSO cell. The antibody molecules prepared as described herein can be purified by known conventional techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and are known to those of skill in the art. The purity of the antibody molecules of the present invention can be determined by any of a variety of known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc. The physical / chemical properties and / or biological activity of the antibody molecules according to the present invention can be identified, selected or otherwise characterized by a variety of assays known in the art.

[0171] V. Pharmaceutical Compositions, Drug Combinations, and Reagent Kits In one aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising an antibody molecule as described herein formulated with a pharma- ceutically acceptable vector. As used herein, a "pharmaceutically acceptable vector" includes any or all of physiologically compatible solvents, dispersion media, isotonicity agents, absorption delaying agents, and the like. The pharmaceutical composition of the present invention is suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal or epidermal administration (e.g., injection or infusion). In some embodiments, the antibody molecule of the present invention is the only active ingredient in the pharmaceutical composition. In some other embodiments, the pharmaceutical composition may comprise an antibody molecule as described herein and one or more therapeutic agents.

[0172] In another embodiment, the present invention further provides a pharmaceutical combination comprising the antibody molecule described herein and one or more therapeutic agents.

[0173] The therapeutic agent applied in the pharmaceutical composition and drug combination of the present invention may be any one of therapeutic agents selected from the following categories (i) to (iii): (i) drugs that enhance antigen presentation (e.g., tumor antigen presentation), (ii) drugs that enhance effector cell responses (e.g., B cell and / or T cell activation and / or recruitment), (iii) drugs that reduce immunosuppression, and (iv) drugs that have a tumor-suppressing effect.

[0174] The compositions of the present invention can be in various forms. These forms include liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable solutions, infusion solutions), dispersions or suspensions, liposomes and suppositories. The preferred form is determined by the desired mode of administration and therapeutic use. Generally, it is preferred that the composition is in the form of an injectable or infusion solution. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (ip), intramuscular) injection. In a preferred embodiment, the antibody molecule is administered by intravenous infusion or injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal or subcutaneous injection.

[0175] As used herein, the terms "parenteral administration" and "parenteral mode of administration" refer to modes of administration other than enteral and topical administration, typically administered by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intradermal, intraperitoneal, transtracheal, subcutaneous injection, and infusion.

[0176] Therapeutic compositions are generally sterile and stable under the conditions of manufacture and storage. The compositions can be prepared in the form of solutions, microemulsions, dispersions, liposomes, or lyophilisates. Sterile injectable solutions can be prepared by adding the active compound (i.e. antibody molecules) in a given amount to a suitable solvent, followed by filtration and sterilisation. Dispersions are generally prepared by mixing the active compound in a sterile solvent, which contains a basic dispersion medium and other ingredients. A coating agent such as lecithin can be used. In the case of dispersions, surfactants can be used to maintain the proper fluidity of the solution. Absorption of injectable compositions can be prolonged by including in the composition a substance that delays absorption, such as monostearate, gelatin.

[0177] In some embodiments, the antibody molecule of the present invention is administered orally, e.g., orally with an inert diluent or edible vector. The antibody molecule of the present invention may be encapsulated in hard or soft gelatin capsules, compressed into tablets, or simply incorporated into the subject's diet. When used for oral therapy, the compound may be incorporated with excipients and used in the form of ingested tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer the antibody molecule of the present invention via a parenteral route, the antibody molecule may be coated with or administered with a material that prevents its inactivation. Additionally, the therapeutic composition may be administered using medical devices known in the art.

[0178] The pharmaceutical composition of the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of the antibody molecule according to the present invention. A "therapeutically effective amount" refers to an amount to effectively achieve a desired therapeutic result at a required dosage for a required period of time. The therapeutically effective amount varies depending on many factors, such as the disease state, the age, sex, and weight of the individual. A therapeutically effective amount is any amount that does not cause toxicity or harm beyond the beneficial effects of the treatment. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% compared to a subject not receiving the treatment. The ability of the antibody molecule of the present invention to inhibit the measurable parameter (e.g., tumor volume) can be evaluated in an animal model system to show efficacy in human tumors.

[0179] A "prophylactically effective amount" refers to an amount to effectively achieve the desired prophylactic result at the necessary dosage for the necessary period of time. Generally, since a prophylactic dose is used in subjects prior to or at a relatively early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0180] The present invention also includes a reagent kit comprising the antibody molecule described herein.The kit may also include one or more other elements, such as instructions, other reagents such as markers or coupling reagents, pharma-ceutically acceptable vectors, devices or other materials for administering to subjects.

[0181] V. Uses and Methods of the Molecules of the Invention In one aspect, the present invention provides in vivo and in vitro uses and methods of application of the antibody molecules of the present invention.

[0182] In some embodiments, the uses and methods of the present invention relate to the following in vivo and / or in vitro applications of the antibody molecules of the present invention: - binding to the GPRC5D antigen with high affinity, such as a KD of less than 10 nM, comprising expressing the GPRC5D antigen on the surface of a cell; - targeting T cells (e.g. CD4+ and / or CD8+ T cells) to cells expressing GPRC5D on their surface, in particular GPRC5D-positive tumor cells; - targeting T cells (e.g., CD4+ and / or CD8+ T cells) to cells expressing BCMA on their surface, in particular BCMA-positive tumor cells; -activating the CD3 downstream signaling pathway in T cells; - mediating the killing effect of T cells against GPRC5D-positive and / or BCMA-positive tumor cells; -inducing T cells to release cytokines such as TNF-α, IFN-γ, and IL-2; - inhibiting or killing GPRC5D-positive and / or BCMA-positive tumor cells, or - Treating GPRC5D positive and / or BCMA positive multiple myeloma, including treating BCMA positive patient populations, GPRC5D positive patient populations, GPRC5D positive patient populations with BCMA loss following binding to an anti-BCMA molecule, - Avoiding BCMA escape-mediated tumor relapse, such as relapse of multiple myeloma.

[0183] In some embodiments, the antibody molecule of the invention or a pharmaceutical composition comprising the antibody molecule of the invention is used as a medicament for treating and / or preventing a disease or as a diagnostic tool for a disease in an individual, preferably said individual is a mammal, more preferably a human.

[0184] In some embodiments, the present invention provides a method and use of the antibody molecule of the present invention, particularly the trispecific antibody molecule, for the treatment of cancer, wherein the cancer may be selected from multiple myeloma, melanoma, B-cell lymphoma. Preferably, the cancer is multiple myeloma. Due to the antigen-binding specificity for BCMA and GPRC5D, the trispecific antibody molecule of the present invention can have a broader patient population for cancer treatment than the bispecific antibody targeting BCMA and CD3 and the bispecific antibody targeting GPRC5D and CD3. In some embodiments, the present invention provides a method for the treatment of BCMA-negative or BCMA-low expressing cancer using the trispecific antibody molecule of the present invention. In further some embodiments, the present invention provides the treatment of GPRC5D and CD3 double positive cancer using the trispecific antibody molecule of the present invention.

[0185] In one aspect, the invention provides a diagnostic method for detecting the presence of a relevant antigen in a biological sample, such as serum, semen, urine or a tissue biopsy sample (e.g., from a hyperproliferative or cancerous lesion), in vitro or in vivo. The diagnostic method comprises (i) contacting the sample (and optionally a control sample) with an antibody molecule as described herein or administering said antibody to a subject under conditions in which an interaction is permitted to occur, and (ii) detecting the formation of a complex between said antibody and the sample (and optionally the control sample). Formation of a complex indicates the presence of the relevant antigen and also indicates suitability for or need for treatment and / or prevention as described herein.

[0186] In some embodiments, the relevant antigen is detected before treatment, e.g., before treatment is started or, if there is a treatment interval, before treatment is resumed. Available detection methods include immunohistochemistry, immunocytochemistry, FACS, ELISA assays, PCR techniques (e.g., RT-PCR) or in vivo imaging techniques. Generally, the antibody molecules used in in vivo and in vitro detection methods are directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound conjugate. Suitable detectable substances include various biologically active enzymes, prosthetic groups, fluorescent materials, luminescent materials, paramagnetic (e.g., nuclear magnetic resonance active) materials, and radioactive materials.

[0187] In some embodiments, the level and / or distribution of the relevant antigen is determined in vivo, e.g., an antibody molecule of the invention labeled with a detectable substance is detected in a non-invasive manner, e.g., by a suitable imaging technique, e.g., positron emission tomography (PET) scanning. In one embodiment, the level and / or distribution of the relevant antigen is measured in vivo, e.g., by detecting an antibody molecule of the invention detectably labeled with a PET reagent, e.g., 18F-fluorodeoxyglucose (FDG). In one embodiment, the invention provides a diagnostic kit comprising an antibody molecule described herein and instructions for use.

[0188] The following examples are presented to aid in the understanding of the present invention, and are not intended, and should not be construed, as limiting the scope of the invention in any manner whatsoever. EXAMPLES

[0189] Working Example Example 1. Preparation of hybridoma cells Construction of overexpression cell lines The full-length sequences of human, monkey and mouse GPRC5D were inserted into the PEE17.4 plasmid (Lonza, GS Xceed Expression System), the plasmid was introduced into the host cell GS-CHO by electroporation, and a stable cell line overexpressing GPRC5D protein was constructed by pressure screening. At the same time, the full-length sequence of human GPRC5D was inserted into the pCHO1.0 vector and introduced into the host cell 293F to construct the overexpression cell line 293F-huGPRC5D.

[0190] immunity The full-length sequence of human GPRC5D was constructed in pcDNA3.1 vector, and the plasmid was used to immunize Balbc and Harbour mice (purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) by intramuscular injection once every two weeks (50 μg of plasmid for each mouse), for a total of three immunizations. Then, the GS-CHO cell line overexpressing human GPRC5D was used for boosting, by intraperitoneal injection once every two weeks (1 × 10 7 ), and were immunized a total of two times.

[0191] Cell fusion After the serum titer met the requirement, the mouse spleen was removed to prepare a B lymphocyte suspension, which was then mixed with SP2 / 0 myeloma cells (ATCC) at a ratio of 1:2 to 1:1 and then subjected to electric fusion. The fused cells were transferred from the electrode dish to a 50 mL centrifuge tube and diluted to 1 × 10 in HAT medium. 4 Cells / mL ~ 2 x 10 4 The cells were diluted to cells / mL, and 100 μL of the cell suspension was added to each well of a 96-well plate. The screening medium was replaced on the 7th day after fusion, and positive cloning was screened by flow cytometry (FACS) detection after 10 days (or longer depending on the cell growth state) of culture.

[0192] High-throughput screening with hybridoma cells Hybridoma cells specifically expressing anti-GPRC5D antibodies were screened by flow cytometry (FACS). The detected cells (293F-huGPRC5D / GS-CHO-huGPRC5D) were counted and 1 × 10 6 The cells were diluted to 100 cells / mL and added to a U-bottom 96-well plate at 100 μL / well. Centrifuged at 500 g for 5 min and the cell culture medium was removed. The supernatants from the hybridomas cultured in the 96-well plate and the positive control antibody were added to the U-bottom plate and the cells were resuspended, added at 100 μL per well, and left on ice for 30 min. Centrifuged at 500 g for 5 min, the supernatant was removed, and the cells were washed once with PBS. Centrifuged at 500 g for 5 min and the PBS was removed. 100 μL of goat anti-mouse IgG FITC-labeled secondary antibody (Jackson Immunoresearch, Cat#115-545-006, diluted 1:500 in PBS) was added to each well containing hybridoma supernatant, and 100 μL of anti-human Fc PE-labeled secondary antibody (BioLegend, Cat#409304) was added to wells containing positive control antibody (Roche-5E11). Incubate on ice for 30 min protected from light. Remove supernatant and wash cells once with PBS at 500g for 5 min. Resuspend cells in 50 μL of 1×PBS and perform detection on FACS machine.

[0193] The screened positive clones were subjected to rescreening of GS-cynoGPRC5D / GS-huGPRC5A cells by the same method as above, and a total of 82 hybridoma cell lines were obtained that bound to both human GPRC5D and monkey GPRC5D but not to human GPRC5A.

[0194] Subcloning of positive hybridoma cells Based on the results of the cell binding experiment, the candidate clones were subcloned by conventional methods, and then detected by the above-mentioned high-throughput screening method, and the target-positive wells were picked up and the cells were cryopreserved.

[0195] Example 2: Preparation of chimeric antibodies The gene sequences of the antibody light and heavy chains were extracted from the hybridoma candidate clones obtained in Example 1, and a human-mouse chimeric antibody was constructed.

[0196] Freshly cultured cells were cultured at approximately 5 × 10 6 The samples were collected and RNA (Macherey-Nagel, Cat#740984.250) was extracted. cDNA was obtained by reverse transcription using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara). An upstream primer was designed with a base sequence located in the 5'-terminal FR1 region, and a downstream primer was designed with a base located in the antibody constant region or FR4 region to amplify the variable region gene fragments of the antibody light and heavy chains. The fragments were ligated into a T vector (Mighty TA-cloning Kit, Takara), and monoclonals were selected and sequenced. Analysis and alignment were performed against the sequencing results using MEGA7 software.

[0197] Through alignment, the antibody light chain and heavy chain variable region sequences were selected for correct and paired cloning, and the light chain and heavy chain variable region gene fragments were linked to the pcDNA3.1 vector respectively using the homologous recombination enzyme (Exnase® II, catalog number: C112-01) from Nanjing Nuova Corporation, where the IgG1 subtype was selected for the constant region, and expression plasmids for light chain and heavy chain antibodies were obtained.

[0198] Next, the light chain plasmid and heavy chain plasmid of the same antibody were mixed at a molar ratio of 1:1 and transfected into 293F cells using polyethyleneimine (PEI) (Polysciences, Cat#23966). After culturing for 5 to 7 days, when the cell activity was less than 60%, the cell culture supernatant was collected and the monoclonal antibody was purified using a Protein A affinity column.

[0199] Example 3. In vitro screening of chimeric antibodies The affinity of anti-GPRC5D antibodies was detected by flow cytometry. Different concentrations of antibodies were co-incubated with cells expressing human GPRC5D, including GS-CHO cells overexpressing human GPRC5D (huGPRC5D GS-CHO), multiple myeloma cells MM1.R, H929 and AMO-1 for 30 minutes. Then, fluorescently labeled secondary antibodies (APC-mouse anti-human IgG Fc antibody, Biolegend, Product No.: 409306) were added, and the cell fluorescence intensity was detected by flow cytometry using Live / Dead yellow dead cell stain (Thermo Fisher, Product No.: L34967). The curves were fitted using GraphPad Prism 8.0, and the EC50 value was calculated to indicate the affinity of the antibodies to bind to human GPRC5D.

[0200] Among the detected antibodies, HB15H1G1 (heavy chain variable region SEQ ID NO: 25, light chain variable region SEQ ID NO: 26) is a fully humanized antibody, and ch5E12C4 (heavy chain variable region SEQ ID NO: 98, light chain variable region SEQ ID NO: 99), ch11B7 (heavy chain variable region SEQ ID NO: 100, light chain variable region SEQ ID NO: 101) and ch7F5D4 antibodies (heavy chain variable region SEQ ID NO: 27, light chain variable region SEQ ID NO: 28) are chimeric antibodies. The results are shown in Table 1 below. [Table 4]

[0201] The screened ch5E12C4 and chimeric ch11B7 antibodies were humanized to form antibodies hz5E12.1.P1 and hz11B7.5.

[0202] Example 4: Design and construction of the molecular structure of trispecific antibodies (TS) Multispecific antibodies of the T-cell engager class that simultaneously target GPRC5D, BCMA, and CD3 can simultaneously bind to two tumor-associated antigens, GPRC5D and BCMA, on the surface of multiple myeloma (MM) cells, and to the CD3 receptor on the surface of T cells. We designed multispecific antibodies that simultaneously target GPRC5D, BCMA, and CD3 based on four anti-GPRC5D antibodies (HB15H1B1, ch7F5D4, hz5E12.1.P1, hz11B7.5), one anti-BCMA antibody (ADI-38456), and two anti-CD3 antibodies with different affinities (CD3 antibody hzsp34.87 with a relatively low affinity and CD3 antibody hzsp34.24 with a relatively high affinity).

[0203] As shown in Figure 1, four types of multispecific antibody molecules with different formats were designed, including two exemplary antibodies in 1+1+1 format 1 (TS-F1), six exemplary antibodies in 1+1+1 format 2 (TS-F2), four exemplary antibodies in 2+2+2 format 6 (TS-F6), and five exemplary antibodies in 1+1+2 format 7 (TS-F7) (see Table 4 below). The mispairing of the heavy chains of asymmetric IgG-like bispecific antibodies was resolved by Fc "knob-in-hole" technology, where Fc is the heavy chain constant region of IgG1, and the exemplary antibodies were designed by introducing amino acid mutations L234A, L235A (numbered by "EU" in Kabat) that simultaneously weaken the effector function.

[0204] TS-F2 and TS-F7 were composed of asymmetric IgG-like tetramers consisting of four polypeptide chains, whereas TS-F6 was composed of a symmetric IgG-like tetramer consisting of two polypeptide chains.

[0205] TS-F2 was composed of one peptide chain #1 containing a heavy chain variable domain, an immunoglobulin CH1 domain, and an Fc domain, one peptide chain #2 containing a light chain variable domain and an immunoglobulin CL domain, one peptide chain #3 containing a single-chain antibody scFv linked by a heavy chain variable domain, an immunoglobulin CH1 domain, an Fc domain, and an artificially synthesized linking peptide, and one peptide chain #4 containing a light chain variable domain and an immunoglobulin CL domain. The heavy chain variable domain of peptide chain #1 and the light chain variable domain of peptide chain #2 were paired to form the first antigen recognition site, the heavy chain variable domain of peptide chain #3 and the light chain variable domain of peptide chain #4 were paired to form the second antigen recognition site, and the scFv formed the third antigen recognition site.

[0206] TS-F7 was composed of one peptide chain #1 containing a heavy chain variable domain, an immunoglobulin CH1 domain and an Fc domain, one peptide chain #2 containing a light chain variable domain, an immunoglobulin CL domain and a single chain antibody scFv linked by an artificially synthesized linking peptide, one peptide chain #3 containing a heavy chain variable domain, an immunoglobulin CH1 domain and an Fc domain, and one peptide chain #4 containing a light chain variable domain, an immunoglobulin CL domain and a single chain antibody scFv linked by an artificially synthesized linking peptide. The heavy chain variable domain of peptide chain #1 and the light chain variable domain of peptide chain #2 are paired to form a first antigen recognition site, the heavy chain variable domain of peptide chain #3 and the light chain variable domain of peptide chain #4 are paired to form a second antigen recognition site, and the two scFvs in peptide chain #2 and peptide chain #4 are the two same third antigen recognition sites.

[0207] In TS-F2 and TS-F7, the Fc domains of peptide chain #1 and peptide chain #3 contained the corresponding stabilizing binding mutations of “knobs-in-holes,” respectively.

[0208] TS-F6 was composed of two identical peptide chains #1 containing a heavy chain variable domain, an immunoglobulin CH1 domain, an Fc domain, and a single-chain antibody scFv linked by an artificially synthesized linking peptide, and two identical peptide chains #2 containing a light chain variable domain, an immunoglobulin CL domain, and a single-chain antibody scFv linked by an artificially synthesized linking peptide. The heavy chain variable domain of peptide chain #1 and the light chain variable domain of peptide chain #2 are paired to form a first antigen recognition site, the scFv in peptide chain #1 is the second antigen recognition site, and the scFv in peptide chain #2 is the third antigen recognition site.

[0209] TS-F1 was composed of peptide chain #1 containing a first single-chain antibody scFv and an Fc domain, and peptide chain #2 containing a second single-chain antibody scFv and an Fc domain. The first scFv in peptide chain #1 formed the first antigen recognition site, and the scFv in peptide chain #2 formed the second antigen recognition site.

[0210] Example 5. Expression and purification of TS antibodies Of the four anti-GPRC5D antibodies (HB15H1B1, ch7F5D4, hz5E12.1.P1, hz11B7.5), HB15H1B1 is a fully humanized antibody, ch7F5D4 is a chimeric antibody, and hz5E12.1.P1 and hz11B7.5 are humanized antibodies. The amino acid sequences of the CDR regions, light chain variable regions and heavy chain variable regions of one anti-BCMA antibody (ADI-38456) and two anti-CD3 antibodies with different affinities (hzsp34.87 and hzsp34.24) are listed in the "Sequence Listing" section of this application, and the sequence numbers of the amino acid sequences of the CDR regions, light chain variable regions and heavy chain variable regions of the antibodies are shown in Table 2. [Table 5] [Table 6] [Table 7] [Table 8]

[0211] GPRC5DxCD3 and BCMAxCD3 bispecific antibodies were designed simultaneously. As shown in Figure 2A, the bispecific antibodies were rationally designed with both ends in Fab format, forming an asymmetric Ig-like structure in 1+1 format. The mispairing of the heavy chains of the asymmetric IgG-like bispecific antibodies was resolved by Fc "knob-in-hole" technology, where Fc is the heavy chain constant region of IgG1, and amino acid mutations L234A and L235A (numbered by Kabat "EU") were introduced to weaken the effector function.

[0212] Concurrently, a BCMAxCD3 bispecific antibody 46758 was further expressed. As shown in FIG. 2B, one antibody arm of the bispecific antibody is a Fab that binds BCMA and the other antibody arm is an scFv that binds CD3, where the Fab is derived from the anti-BCMA parent antibody 38456 and the scFv is derived from a different anti-CD3 parent antibody. The three polypeptide chains that made up the bispecific antibody are shown in SEQ ID NOs: 111-113.

[0213] At the same time, the Roche-5E11 bispecific antibody (GPRC5DxCD3, 2:1 format, from patent WO 2019 / 154890 A1) was further expressed and purified, as shown in Figure 2C. The bispecific antibody Roche_5E11 is composed of the sequences of SEQ ID NOs: 17, 18, 19 and 20 of WO 2019 / 154890 A1, and contains two GPRC5D binding sites and one CD3 binding site.

[0214] For recombinant production, we commissioned Suzhou Jinweizhi Biotechnology Co., Ltd. to synthesize the nucleotide sequences encoding the heavy and light chains and insert them into the vector pcDNA3.1, respectively, for further cloning and / or expression in host cells.

[0215] Expression in HEK293 cells and purification: Expi293F cells (purchased from Thermo Fisher Scientific) were subcultured in Expi293F cell medium (purchased from Thermo Fisher Scientific). The day before transfection, the cell density was detected and fresh Expi293 cell medium was added at 2 × 10 6 Continue culturing by adjusting the cell density to 3 × 10 cells / mL on the day of transfection. 6 The concentration was adjusted to cells / mL.

[0216] The final volume of the transfected Expi293F cells was 1 / 10 of Opti-MEM medium (purchased from Gibco) as a transfection buffer, and 10 μg of the recombinant plasmid of the nucleotide sequence containing the paired heavy chain and light chain prepared above was added to each 1 mL of transfection buffer at a molar ratio of 1:1, mixed evenly, and 30 μg of polyethyleneimine (PEI) (Polysciences) was further added to each mL of transfection buffer, mixed evenly, and incubated at room temperature for 20 minutes, and then the PEI / DNA mixture was gently injected into the Expi293F cell suspension to mix evenly, and cultured in a shaker under the culture conditions of 8% CO2, 36.5°C, and 120 rpm.

[0217] After culturing for 16 to 18 hours, 200g / L of FEED (100g / L of Phytone Peptone + 100g / L of Difco Select Phytone) was added to the culture flask at a concentration of 1 / 50 of the culture volume after transfection, a glucose solution at a final concentration of 4g / L, and VPA (Gibco) at a final concentration of 2mM / L were added, mixed gently and homogenously, and the culture was continued in a shaker at 8% CO2, 36.5℃, and 120 rpm. After continuous culturing for 6 days, the culture was collected and centrifuged at 4000 rpm for 30 minutes, the cell supernatant was taken and filtered through a 0.45μM filtration membrane, and the parent protein was purified by affinity chromatography and ion exchange chromatography. For symmetrical IgG-like antibodies, the final molecules were purified.

[0218] The recombinantly produced parent proteins for the above-mentioned TS antibodies and bispecific antibodies in different formats are shown below. [Table 9]

[0219] In the case of asymmetric IgG-like antibodies, the two purified parent proteins do not need to exchange solutions, and the protein concentrations are >1mg / mL each, and mixed according to a 1:1 molar ratio and protein concentration in the mixed solution of 1mg / mL-10mg / mL. Then, the molar ratio of GSH / protein is controlled to 500-700 times, and an appropriate amount of GSH is added, with a GSH final concentration of >5mM. Finally, the pH of the mixed solution is adjusted to 8.0-8.5 with Arg1M at pH 10.0. The Arg final concentration is >=50mM. The reaction solution is left at room temperature overnight, not exceeding 24h. The next day, the reaction solution that has been reacted overnight is exchanged to remove GSH, and the buffer for liquid exchange may be the recombinant reaction 0.2M PB solution (Ph6.0) without GSH. After natural oxidation for 2h-3h, it may be purified by monoS (GE Cat.17516801).

[0220] The Roche-5E11 bispecific antibody was produced by the method disclosed in WO 2019 / 154890 A1.

[0221] The symmetric and asymmetric antibodies produced by the above method were collected and purified by affinity chromatography and ion exchange chromatography. Size exclusion chromatography (SEC) was used to detect the purity of the samples in the sub-tubes of each level collected by chromatography. Samples in the sub-tubes with a purity of more than 95% based on the SEC results were combined. The purified bispecific antibody solution was centrifuged in a 15 mL ultrafiltration centrifuge tube at 4500 rpm for 30 minutes, and the centrifugation was continued after diluting the protein with PBS, and the centrifugation was repeated for 30 minutes at 4500 rpm to exchange the buffer solution, and the above operation was repeated several times. The antibodies with exchanged buffer solution were combined to measure the antibody concentration. Furthermore, the components and contents of the multispecific antibody were qualitatively and quantitatively analyzed by combining capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0222] Example 6. Measurement of trispecific antibody affinity In the exemplary trispecific antibody, the binding affinity of the antigen-binding site derived from humanized antibody hz5E12.1.P1 and hz11B7.5 to GPRC5D was examined. By the same method as in Example 3, different concentrations of the trispecific antibody were co-incubated with huGPRC5D GS-CHO cells for 30 minutes, then fluorescently labeled secondary antibody was added, and the cell fluorescence intensity was detected by flow cytometry (BD), and the curve was fitted using GraphPad Prism 8.0, and the EC50 value was calculated to show the affinity of the antibody to bind to human GPRC5D. The results are shown in Table 5 below. [Table 10]

[0223] In the exemplary trispecific antibody, the affinity of the anti-BCMA and anti-CD3 termini was detected by biofilm layer optical interference technology (BLI). The binding kinetics of the exemplary antibodies to human BCMA, monkey BCMA, human CD3E and monkey CD3E were measured.

[0224] Conjugated streptavidin protein (SA) or anti-human-Fc (AHC) probe sensors (Fortebio, product number 18-5019) were pre-wetted by immersion in 200 μL of SD buffer (1×PBS, 0.1% BSA, 0.05% tween-20). Exemplary antibodies and biotin-labeled or Fc-tagged BCMA and CD3D&E heterodimer antigens were diluted in SD buffer, respectively. 200 μL of SD buffer, each diluted sample (100 nM) was added to a 96-well black plate (Greiner, product number 655209), respectively. The probes and samples were placed in Octet (Fortebio, Red96e). Data Acquisition 10.0 was launched, "New Kinetic Experiment" was selected, the samples were seeded according to their positions, the position of the sensors was selected, and the execution steps and times were set as follows: Baseline 60s, Loading 250s, Baseline 100s, Association 600s, and Dissociation 600s. The rotation speed of the experiment was 1000 rpm, and the temperature was 30°C.

[0225] Results were analyzed with "Data Analysis 10.0" software to subtract the buffer reference channel, select 1:1 binding to fit the data, and calculate k on , k off , and K D values ​​for exemplary antibodies.

[0226] In the experiments described in the above assays, exemplary antibodies were detected using human BCMA antigen (ACRO Biosystems) and monkey BCMA antigen (ACRO Biosystems). The results of the detection of affinity to human BCMA are shown in Table 6, and the results of the detection of affinity to monkey BCMA are shown in Table 7. [Table 11] [Table 12]

[0227] In the experiments described in the above assay, the affinity of the exemplary antibodies was detected using human CD3D&E heterodimeric antigen (ACRO Biosystems) and monkey CD3D&E heterodimeric antigen (ACRO Biosystems), and the results are shown in Tables 8 and 9. In parallel, the binding affinity of GPRC5DxCD3 bispecific antibodies, hz5E12.1-P1 / SP34.24 and hz5E12.1-P1 / SP34.87 to human / monkey CD3D&E was examined. [Table 13] [Table 14]

[0228] Example 7: Anti-GPRC5DxBCMAxCD3 antibody-mediated Jurkat cell activation experiment The activity of anti-GPRC5DxBCMAxCD3 antibody to activate CD3E downstream signaling pathway was detected by Jurkat-NFAT-Luc report system. When anti-GPRC5DxBCMAxCD3 multiple antibody binds to GPRC5D and / or BCMA on the surface of multiple myeloma (MM) cell line and simultaneously binds to CD3E on the surface of Jurkat-NFAT-Luc cells (Jurkat), it can stimulate CD3E downstream signal in Jurkat-NFAT-Luc cells by crosslinking with tumor-associated antigen (GPRC5D and / or BCMA)-dependent CD3. Therefore, the exemplary antibody-mediated T cell activation level was evaluated by Luciferase report system.

[0229] Different concentrations of exemplary antibodies were added in Jurkat NFAT luciferase cells and MM tumor cells, and the expression of Jurkat cell luciferase was observed. [Table 15]

[0230] Briefly, first, sample dilution: the antibody was diluted to a concentration of 100 nM using RPMI medium 1640 (5% FBS), followed by 5-fold gradient dilution. Next, cell preparation: Jurkat cells and tumor cells were centrifuged at 300 g for 5 min, the supernatant was discarded, and the cells were resuspended in RPMI medium 1640 (5% FBS). After counting (Countstar), the cell density was Jurkat NFAT 2x10 6The concentration of each well was adjusted to 100 μL / mL, and the tumor cells were adjusted according to the ratio of effector cells to target cells. 45 μL of tumor cells were added to each well of a 96-well white cell culture plate, 30 μL of the gradient diluted sample was added, and 45 μL of Jurkat NFAT cells were added. The 96-well plate was then placed in a 37°C, 5% CO2 incubator to continue the culture. After a certain period of culture, the culture plate and cell culture plate were removed and placed at room temperature for 5 min, 80 μL of Bio-Glo (Promega, G7940) was added to each well, incubated in the dark for 10 min, and the fluorescence value was read using SpectraMax i3 (MOLECULAR DEVICES). The curves were fitted using GraphPad Prism 8.0, and the EC50 value was calculated to compare the activity of the exemplary antibodies to activate T cells. The results are shown as the fold ratio of the experimental group reading to the control hIgG1 group reading.

[0231] result: TS-F2-1 and TS-F2-2 Under the condition of co-culture of H929 cells and Jurkat cells (effector cell to tumor cell ratio of 5:1) for 16 hours, and under the condition of co-culture of L363 cells and Jurkat cells (effector cell to tumor cell ratio of 5:1) for 16 hours, TS-F2-1 and TS-F2-2 can significantly improve the expression of Jurkat cell luciferase with increasing antibody concentration (Figure 3A and Figure 3B), and their agonist activity is higher than that of TS-F1, control GPRC5DxCD3 double antibody and BCMAxCD3 double antibody. The specific EC50 of the detected antibodies is shown in Table 10. Here, exemplary antibodies TS-F2-1 and TS-F2-2 are both composed of three binding specificities derived from HB15H1G1 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody), but the positions of the binding specificities of HB15H1G1 and 38456 are different. [Table 16]

[0232] TS-F2-3 Under the conditions of co-culture of H929 cells and Jurkat cells (effector cell to tumor cell ratio of 5:1) for 16 hours, co-culture of L363 cells and Jurkat cells (effector cell to tumor cell ratio of 5:1) for 16 hours, and co-culture of U266 cells and Jurkat cells (effector cell to tumor cell ratio of 5:1) for 16 hours, TS-F2-3 can significantly improve the expression of Jurkat cell luciferase with increasing antibody concentration (Figure 4A, Figure 4B and Figure 4C), and its agonist activity is significantly higher than that of TS-F6 antibody, and higher than that of the control group GPRC5DxCD3 double antibody (ch7F5D4 / hzsp34.24) and BCMAxCD3 (46758) double antibody. The specific EC50 of the detected antibodies is shown in Table 11. Exemplary antibodies TS-F2-3 and TS-F6 (TS-F6-1 to TS-F6-4) are composed of three binding specificities derived from ch7F5D4 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody).

[0233] The constructed huGPRC5D-overexpressing GS-CHO (huGPRC5D-GS-CHO) cells were co-cultured with Jurkat cells for 16 hours, and the exemplary antibodies were co-cultured to simulate multiple myeloma cell-mediated T cell activation mediated by GPRC5D-only expressing multiple myeloma cells. Here, the ratio of effector cells (Jurkat cells) to target cells (huGPRC5D-GS-CHO cells) was 50:1. The experimental results showed that TS-F2-3 could significantly improve the expression of Jurkat cell luciferase with increasing antibody concentration (Figure 4D), and its agonist activity was higher than that of TS-F6 antibody, control GPRC5DxCD3 double antibody and BCMAxCD3 double antibody (46758). The specific EC50 is shown in Table 11. [Table 17]

[0234] TS-F2-4 and TS-F2-5 H929 cells and Jurkat cells (effector cell to tumor cell ratio 5:1) were co-cultured for 5 hours. L363 cells and Jurkat cells (effector cell to tumor cell ratio 5:1) were co-cultured for 5 hours. 8226 cells were co-cultured with Jurkat cells (effector cell to tumor cell ratio of 5:1) for 5 h, and U266 cells and Jurkat cells (effector cell to tumor cell ratio 5:1) were co-cultured for 5 hours. Both TS-F2-4 and TS-F2-5 can significantly improve the expression of Jurkat cell luciferase with increasing antibody concentration (FIG. 5A, FIG. 5B, FIG. 5C), and their agonist activity is higher than that of the control groups GPRC5DxCD3, BCMAxCD3 double antibody (38456 / hzsp34.24) and 46758 double antibody. Specific EC50 is shown in Table 12. Here, the exemplary antibody TS-F2-4 is composed of hz5E12.1.P1 (GPRC5D antibody), hzsp34.24 (high affinity CD3 antibody) and 38456 (BCMA antibody), and the exemplary antibody TS-F2-5 is composed of hz5E12.1.P1 (GPRC5D antibody), hzsp34.87 (low affinity CD3 antibody) and 38456 (BCMA antibody). [Table 18]

[0235] Under the condition of co-culture of MM1.S cells and Jurkat cells (effector cell to tumor cell ratio of 10:1) for 5 hours, and under the condition of co-culture of ARD cells and Jurkat cells (effector cell to tumor cell ratio of 10:1) for 5 hours, TS-F2-4 and TS-F2-5 can significantly improve the expression of Jurkat cell luciferase with increasing antibody concentration (Figure 5E), and their agonist activity is higher than that of the control group GPRC5DxCD3 double antibody and BCMAxCD3 double antibody 46758. Specific EC50 is shown in Table 13. [Table 19]

[0236] TS-F7-1 and TS-F7-2 H929 cells and Jurkat cells (effector cell to tumor cell ratio 5:1) were co-cultured for 5 hours. L363 cells and Jurkat cells (effector cell to tumor cell ratio 5:1) were co-cultured for 5 hours. 8226 cells were co-cultured with Jurkat cells (effector cell to tumor cell ratio of 5:1) for 5 h, and U266 cells and Jurkat cells (effector cell to tumor cell ratio 5:1) were co-cultured for 5 hours. TS-F7-1 and TS-F7-2 can significantly improve the expression of Jurkat cell luciferase with increasing antibody concentration (FIGS. 6A, 6B, 6C, 6D), and their agonistic activity is higher than that of the control GPRC5DxCD3 double antibody and BCMAxCD3 double antibody 46758. Specific EC50 is shown in Table 14. Here, the exemplary antibody TS-F7-1 is composed of hz5E12.1.P1 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody), and the exemplary antibody TS-F7-2 is composed of hz11B7.5 (GPRC5D antibody), hzsp34.87 (CD3 antibody) and 38456 (BCMA antibody). [Table 20]

[0237] TS-F7-3 and TS-F7-4 Under the condition of co-culture of L363 cells and Jurkat cells (effector cell to tumor cell ratio of 5:1) for 16 hours, TS-F7-3 and TS-F7-4 can significantly improve the expression of Jurkat cell luciferase with increasing antibody concentration (Figure 7), but their agonist activity is lower than that of TS-F2-3. Here, exemplary antibodies TS-F7-3 and TS-F7-4 are composed of ch7F5D4 (GPRC5D antibody), hzsp34.24 (CD3 antibody) and 38456 (BCMA antibody), and when the hzsp34.24 antibody forms a single chain antibody, TS-F7-3 has VL in front and VH in back, and TS-F7-4 has VH in front and VL in back.

[0238] Example 8: Killing experiment of anti-GPRC5DxBCMAxCD3 antibody against human multiple myeloma cells Exemplary antibody-mediated T cell killing of human multiple myeloma cells was detected by lactate dehydrogenase (LDH) method under the condition of co-culture of PBMC with GPRC5D and / or BCMA positive MM cells. When anti-GPRC5DxBCMAxCD3 multiple antibody binds to GPRC5D and / or BCMA on the surface of MM cells and simultaneously binds to CD3E on the surface of primary T cells, it can crosslink tumor-associated antigen (GPRC5D and / or BCMA)-dependent T cells to stimulate T cell activation and mediate tumor cell killing. The levels of multiple cytokines were simultaneously detected using a multifactor detection reagent kit (Human Th1 / Th2 / Th17, BD), and the percentage of CD69 positive cells in T cells was detected by flow cytometry (BD).

[0239] The following reagents and materials were used: [Table 21]

[0240] Experimental steps: PBMC cells were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, added to 9 mL of serum-free medium, centrifuged at 300 g for 4 min, the supernatant discarded, and resuspended in 10% FBS phenol red-free 1640 medium to a cell density of 4 × 10 6 An aliquot of log-phase H929 cells was harvested, centrifuged, and cultured in 10% FBS phenol red-free 1640 medium at a tumor cell density of 2 × 10 5 The concentration was adjusted to 10 cells / mL. According to the above table, the antibodies were diluted to 100 nM in 10% FBS phenol red-free 1640 medium, so as to make a 5-fold gradient dilution from 5 μM. 100 μL of tumor cells, 50 μL of the gradient diluted samples, and 50 μL of PBMC cells were added to a 96-well cell culture plate (U-shaped). At the same time, control wells were set up and supplemented with medium to 200 μL / well. The plates were cultured for 16 h at 37°C in a CO2 incubator. After 16 h of culture, the cell culture plate was removed and left at room temperature for 5 min, and 10% Lysis Solution was added to each well of the TM well and lysed for 15 min in the dark. The cell culture plate was centrifuged at 400 g for 5 min, and 50 μL of the supernatant was taken and transferred to a new flat-bottom 96-well plate. 50 μL of LDH color development reagent was added, and the color was developed at room temperature for 30 min in the dark, and 50 μL of stop solution was added. The absorbance was read on a plate reader, and the killing was calculated by the absorbance. An additional 90 μL of the supernatant was taken into a new 96-well plate (V-shaped) for subsequent cytokine detection. Each well of the cell culture plate was washed once with 200 μL of FACS buffer, and the liquid was removed after centrifugation. 45 μL of staining solution (prepared with FACS buffer) was added to each well, and incubated at 4°C for 20 min in the dark, washed once with 200 μL of FACS buffer, and resuspended in 150 μL of FACS buffer before on-board detection.

[0241] Detection of cytokines by CBA: Standards were dissolved in 2mL of assay diluent to become standard 0 (std 0), then 8 wells std 1-8 were diluted 2-fold gradient, and std 9 was the assay diluent blank control. Each bead was vortexed for 5s-10s, 322μL was drawn out, then 2254μL of assay diluent was added, vortexed to mix uniformly, and a 96-well V-shaped plate was prepared, 50μL of uniformly mixed beads were added to each well, 30μL of assay diluent was added to each well, and then 30μL of sample was added to each well, i.e., all samples were diluted 2-fold. PE detection reagent:assay diluent was used after dilution 1:1. 50μL of standard or sample and PE detection reagent were added to the wells, and the plate was sealed with parafilm and covered with aluminum foil. The plate was shaken at room temperature for 5min on a plate shaker at a rotation speed of 1100rpm. Next, the cells were incubated at room temperature for 3 hours. After the incubation, 120 μL of wash buffer was added to each well, and the cells were centrifuged at 300 g for 5 minutes, and the supernatant was aspirated. 120 μL of wash buffer was added to resuspend the cells, and the cells were detected by flow cytometry.

[0242] Test Results: As detected by flow cytometry, the exemplary antibodies were found to be able to activate primary T cells in a dose-dependent manner (FIGS. 8A and 8B), and there was a correlation between the degree of T cell activation and CD3 affinity: the higher the affinity of anti-CD3, the stronger the ability to activate T cells, thereby mediating the killing of PBMCs against H929 cells (FIG. 9), and the release levels of various cytokines accompanying the killing process are shown in FIGS. 11A-11D.

[0243] Example 9. Antitumor efficacy of exemplary antibodies in an H929 tumor-bearing humanized mouse model in vivo Female NOG mice (35-41 days old) were purchased from Beijing Weitongda Laboratory Animal Technology Co., Ltd. The grade was SPF. After receiving the mice, they were acclimated and isolated for 7 days before the start of the study. H929 cells were routinely subcultured for subsequent in vivo experiments, and the cells were collected by centrifugation and dispersed in PBS. 5 × 10 6 Shaved H929 cells were inoculated into the right dorsal flank of NOG mice at 200 μL / cell inoculation volume per mouse. Five days after H929 cell inoculation, 5 × 10 6 Mice were injected intravenously with PBMC cells at 200 μL / cell / mouse inoculation volume.

[0244] Administration: Three days after PBMC cell inoculation, mice were divided into groups (7 mice per group) according to tumor volume and administered the drug. The mice were administered once every 7 days, a total of three times. The tumor volume and body weight of the mice were monitored twice a week. Measurement of tumor volume: The maximum long axis (L) and maximum short axis (W) of the tumor were measured with a caliper, and the tumor volume was calculated according to the following formula: V = L × W 2 Body weight was measured using an electronic balance. Throughout the study, mice were euthanized when the tumor reached the endpoint or when the mouse body weight loss was >20%. Tumor size was calculated.

[0245] Experimental Results The tumor growth curves are shown in Figure 12, and the exemplary antibodies can significantly inhibit the growth of H929 cells. No weight loss was observed in any of the mice treated at the same time.

[0246] Example 10. Expression levels of GPRC5D and BCMA on the surface of different multiple myelomas MM cells were stained with saturating concentrations of BCMA and GPRC5D antibodies using the qifikit reagent kit, and BCMA and GPRC5D molecules on the surface of different multiple myeloma cell lines were quantified by flow cytometry. [Table 22]

[0247] 1×10 5 Different MM cells were added to a 96-well plate, with GS-CHO-huGPRC5D and GS-CHO-huBCMA cells as positive controls and negative controls for each other. Centrifuged at 400g for 5 min, and the supernatant was removed. 10 μL of mouse-derived primary antibodies (BCMA and Mgprc5d-17G1B8) were added and incubated at 4°C for 1 h. Negative control IgG1. QIFIKIT / Beads preparation: 100 μL of uniformly mixed beads (Vial 1 and Vial 2) were added, respectively. 200 μL of FACS buffer was added, washed three times, and the supernatant was removed. 100 μL of secondary antibody (FITC Conjugate, Vial 3 (total 200 μL, diluted 1:50 in 0.01 mol / L PBS) was added and mixed uniformly, and incubated at 4°C for 45 minutes in the dark. 200 μL of FACS buffer was added, washed three times, and then 100 μL of PBS was added, mixed uniformly, and detected by flow cytometry.

[0248] Experimental Results: As shown in FIG. 13, different MM cells tested had different surface GPRC5D and BCMA expression levels. As shown in FIG. 5A-FIG. 5F, FIG. 6A-FIG. 6D and FIG. 7, compared with the control GPRC5D / CD3 bibody and BCMA / CD3 bibody, the evaluated Format 2 and Format 7 trispecific antibodies all induced favorable biological activity in various MM cells (H929, L363, 8226, U266, MM1.S and ARD) with different GPRC5D and BCMA expression levels. This suggests that by targeting GPRC5D and BCMA in combination, the trispecific antibody of the present invention can cover both GPRC5D-positive tumor patients and BCMA-positive tumor patients, improve the coverage rate for patients with multiple tumors, and at the same time avoid tumor recurrence due to the loss of a single antigen such as BCMA, and improve the therapeutic effect.

Claims

1. 1. An antibody or antigen-binding fragment thereof that specifically binds to GPRC5D, comprising: (i) the sequences of the heavy chain variable domains HCDR1, 2 and 3 and the light chain variable domains LCDR1, 2 and 3 as shown in SEQ ID NOs: 13 to 18, respectively; (ii) the sequences of the heavy chain variable domains HCDR1, 2 and 3 and the light chain variable domains LCDR1, 2 and 3 as shown in SEQ ID NOs: 13, 110 and 15 to 18, respectively; (iii) the sequences of the heavy chain variable domains HCDR1, 2 and 3 and the light chain variable domains LCDR1, 2 and 3 as shown in SEQ ID NOs: 1 to 6, respectively; (iv) the sequences of the heavy chain variable domains HCDR1, 2 and 3 and the light chain variable domains LCDR1, 2 and 3 as shown in SEQ ID NOs: 7 to 12, respectively; or (v) the sequences of the heavy chain variable domains HCDR1, 2 and 3 and the light chain variable domains LCDR1, 2 and 3 as shown in SEQ ID NOs: 19 to 24, respectively; An antibody or antigen-binding fragment thereof comprising a combination of CDR sequences selected from the group consisting of:

2. The antibody or antigen-binding fragment thereof is (a) a heavy chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO:29, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence as set forth in SEQ ID NO:30, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (b) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 98 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 99 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; (c) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:25, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (d) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (e) a heavy chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising the amino acid sequence set forth in SEQ ID NO: 32, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; or (f) a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 100, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto, and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 101, or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity thereto; The antibody or antigen-binding fragment thereof of claim 1, comprising a combination of VH and VL amino acid sequences selected from the group consisting of:

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the VH and VL sequences contain cysteine ​​substitutions, for example at position 44 of the VH sequence and position 100 of the VL sequence, thereby forming a disulfide bond between the VH and VL.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding fragment is a Fab, scFv or dsscFv.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody is a monospecific, bispecific or trispecific antibody.

6. The antibody or antigen-binding fragment thereof of claim 5 , wherein the antibody further comprises an antigen-binding site that binds to CD3, or further comprises an antigen-binding site that binds to BCMA and an antigen-binding site that binds to CD3.

7. The antigen-binding site that binds to CD3 is (a) a VH comprising the HCDR1-3 sequences of SEQ ID NOs: 41-43 and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 44-46, respectively; or (b) a VH comprising the HCDR1-3 sequences of SEQ ID NOs: 47, 42, and 43, respectively, and a VL comprising the LCDR1-3 sequences of SEQ ID NOs: 44-46; comprising a VH and a VL selected from the group consisting of Optionally, the antigen-binding site that binds to CD3 comprises a VH and VL amino acid sequence pair selected from the group consisting of SEQ ID NO:48 / 49 and SEQ ID NO:50 / 49; 7. The antibody or antigen-binding fragment thereof of claim 6, further comprising, optionally, when the antigen-binding site that binds to CD3 is a dsscFv, the antigen-binding site further comprises cysteine ​​substitutions introduced into the VH and VL sequences, e.g., at position 44 of the VH sequence and at position 100 of the VL sequence.

8. the antigen-binding site that binds to BCMA comprises a VH comprising HCDR1-3 sequences of SEQ ID NOs: 33-35, respectively, and a VL comprising LCDR1-3 sequences of SEQ ID NOs: 36-38, respectively; Optionally, the antigen binding site that binds to BCMA comprises the VH and VL amino acid sequence pair of SEQ ID NOs: 38 / 39; 7. The antibody or antigen-binding fragment thereof of claim 6, further comprising, optionally, when the antigen-binding site that binds to BCMA is a dsscFv, the antigen-binding site further comprises cysteine ​​substitutions introduced into the VH and VL sequences, such as at position 44 of the VH sequence and at position 100 of the VL sequence.

9. The antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody is a bispecific antibody that binds to GPRC5D and CD3.

10. the antibody is a trispecific antibody comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, the first heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; the first light chain polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable domain VL and an immunoglobulin CL domain; the second heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; and the second light chain polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable domain VL and an immunoglobulin CL domain; wherein the first heavy chain polypeptide chain or the second heavy chain polypeptide chain further comprises an scFv domain conjugated to the C-terminus of the Fc domain, either directly or via a linking peptide; Where: the VH-CH1 of the first heavy chain polypeptide pair and the VL-CL of the first light chain polypeptide pair to form a first antibody arm (M1) that binds to a first antigen; the VH-CH1 of the second heavy polypeptide chain and the VL-CL of the second light polypeptide chain pair to form a second antibody arm (M2) that binds to a second antigen; the Fc domain of the first heavy polypeptide chain and the Fc domain of the second heavy polypeptide chain pair and dimerize to form an antibody stem (Fc::Fc); and the scFv domain conjugated to the C-terminus of the first heavy chain polypeptide chain or the second heavy chain polypeptide chain forms a conjugate (X2) that binds to a third antigen; wherein the first, second and third antigens are different and independently selected from GPRC5D, BCMA and CD3; The antibody or antigen-binding fragment thereof according to any one of claims 6 to 8.

11. the antibody comprises or consists of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, (a) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77 or 78, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; (b) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:65, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:66, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:67, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO:68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; (c) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 69, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 71, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; (d) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 72, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 73, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 74 or 78, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; or (e) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto. The antibody or antigen-binding fragment thereof described in claim 10.

12. the antibody is a trispecific antibody comprising or consisting of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, the first heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; the first light chain polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable domain VL and an immunoglobulin CL domain; the second heavy chain polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable domain VH, an immunoglobulin CH1 domain and an Fc domain; and the second light chain polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable domain VL and an immunoglobulin CL domain; wherein the first light chain polypeptide chain and the second light chain polypeptide chain further comprise an scFv domain conjugated to the C-terminus of the CL domain, either directly or via a linking peptide; Where: the VH-CH1 of the first heavy polypeptide chain and the VL-CL of the first light polypeptide chain pair to form a first antibody arm (M1) that binds to a first antigen; the VH-CH1 of the second heavy polypeptide chain and the VL-CL of the second light polypeptide chain pair to form a second antibody arm (M2) that binds to a second antigen; the Fc domain of the first heavy polypeptide chain and the Fc domain of the second heavy polypeptide chain pair and dimerize to form an antibody stem (Fc::Fc); and the scFv domain conjugated to the C-terminus of the first light chain polypeptide chain and the second light chain polypeptide chain forms a conjugate (X1) that binds to a third antigen; wherein the first, second and third antigens are different and independently selected from GPRC5D, BCMA and CD3; The antibody or antigen-binding fragment thereof according to any one of claims 6 to 8.

13. the antibody comprises or consists of a first heavy chain polypeptide chain, a first light chain polypeptide chain, a second heavy chain polypeptide chain and a second light chain polypeptide chain, (a) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 87 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 88 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77 or 78 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89 or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; (b) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 90, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 91, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; or (c) the first heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the first light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 93 or 96, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; the second heavy chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; and the second light chain polypeptide chain comprises an amino acid sequence of SEQ ID NO: 95 or 97, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99% or more identity thereto; The antibody or antigen-binding fragment thereof described in claim 12.

14. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 13.

15. A vector comprising the polynucleotide of claim 14, preferably an expression vector.

16. 16. A host cell comprising the polynucleotide of claim 14 or the vector of claim 15, optionally being a mammalian cell.

17. 14. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 13, the method comprising culturing a host cell comprising a polynucleotide encoding a polypeptide chain of the antibody under conditions suitable for expressing said polypeptide chain, and assembling said polypeptide chains into the antibody molecule to produce the antibody, under conditions suitable for assembling said polypeptide chains into the antibody molecule.

18. A pharmaceutical composition comprising the antibody molecule of any one of claims 1 to 13 and a pharma- ceutically acceptable carrier.

19. 19. A pharmaceutical composition according to claim 18 for use as a medicament for the treatment and / or prevention of a disease in an individual or as a diagnostic tool for diagnosing a disease.

20. 20. The pharmaceutical composition of claim 19, wherein the disease is cancer, preferably multiple myeloma (MM).

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