Anti-GPRC5DxBCMAxCD3 trispecific antibody and its use

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 triple-specific antigen-binding protein, and more specifically, to a triple-specific antibody that specifically binds to two tumor antigens, GPRC5D and BCMA, and the T cell surface antigen CD3, as well as a pharmaceutical composition containing the same, a method for preparation, and its use. [Background technology]

[0002] T cell bispecific antibodies (BsAbs) are used to treat tumors. Such antibodies can form synapses between cytotoxic T lymphocytes and tumor cells, inducing the destruction of tumor cells. BsAbs typically involve dual targeting of tumor-associated antigens (TAAs) and T cell surface antigens (also known as T cell engagement antigens, or TEAs). However, BsAb-based treatment plans typically depend on the distribution of tumor-associated antigens in the tumor cells being treated. This leads to limitations in treatment selectivity and effectiveness for the patient population. Furthermore, studies have shown that treatments targeting a single TAA site may limit treatment effectiveness because tumor evasion mechanisms can lead to disease recurrence.

[0003] The development of triplicate and / or quadruplicate antibodies has been proposed 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, due to the diverse requirements of various therapeutic products regarding therapeutic functionality and behavior, it has been determined that there is no single "optimal format" applicable to most different desirable 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, effective antibody formats for combining specific target antigens need to be identified by producing and comparing the functionality of different antibody formats. [Overview of the project]

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

[0005] To achieve this objective, the inventors propose 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 the T cell surface antigen (CD3), and by regulating the action of the T cell engager antibody in mediating T cell activation and killing effects, the inventors successfully achieve multi-target combinations and reduce the need for multiple drug combinations. Based on this, Knob-in-Hole technology is further used with an Fc segment to reduce heavy chain mismatch, while the problem of light chain mismatch is resolved by extracellular redox means. The antibodies of the present invention also have good prepareability and good drug discovery potential.

[0006] Accordingly, in one embodiment, the present invention provides a triply specific Y-type antibody molecule having the aforementioned advantages, comprising an antigen-binding site that specifically binds to GPRC5D, BCMA, and CD3, and comprising two antibody arms, a stem located at the C-terminus of the antibody arms, and a complex component compounded at the C-terminus of the antibody arms and / or the stem.

[0007] In a further embodiment, the present invention provides an antibody molecule that specifically binds to GPRC5D, comprising a combination of CDR sequences selected from SEQ ID NOs: 1-6, 7-12, 13-18, or 19-24, 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 a further embodiment, the present invention also provides nucleic acids, vectors, and host cells encoding the antibody molecule of the present invention, as well as methods and uses of the antibody molecule of the present invention, in particular for treating GPRC5D-positive and / or BCMA-positive tumors, such as multiple myeloma. [Brief explanation of the drawing]

[0009] The following preferred embodiments of the invention, described in detail below, will be better understood when read in conjunction with the following drawings. For illustrative purposes, the drawings show the current preferred embodiments of the invention. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings. [Figure 1A] The structures of triplicate antibody molecules designed using Formats 1, 2, 6, and 7 are schematically shown. [Figure 1B] The structures of triplicate antibody molecules designed using Formats 1, 2, 6, and 7 are schematically shown. [Figure 1C] The structures of triplicate antibody molecules designed using Formats 1, 2, 6, and 7 are schematically shown. [Figure 1D] The structures of triplicate antibody molecules designed using Formats 1, 2, 6, and 7 are schematically shown. [Figure 2A] The structure of the control bispecific antibody molecule used in the examples is schematically shown. [Figure 2B] The structure of the control bispecific antibody molecule used in the examples is schematically shown. [Figure 2C] The structure of the control bispecific antibody molecule used in the examples is schematically shown. [Figure 3A] Shows detection of exemplary antibody TS-F2-1 and -2 mediated T cell activation by the Jurkat-NFAT-Luc reporter system. [Figure 3B] Shows detection of exemplary antibody TS-F2-1 and -2 mediated T cell activation by the Jurkat-NFAT-Luc reporter system. [Figure 4A] Shows detection of exemplary antibody TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporter system. [Figure 4B] Shows detection of exemplary antibody TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporter system. [Figure 4C] Shows detection of exemplary antibody TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporter system. [Figure 4D] Shows detection of exemplary antibody TS-F2-3 and TS-F6 mediated T cell activation by the Jurkat-NFAT-Luc reporter system. [Figure 5A] Detection of exemplary antibody TS-F2-4 and 5 mediated T cell activation (A-D) by the Jurkat-NFAT-Luc reporter system, [Figure 5B] Detection of exemplary antibody TS-F2-4 and 5 mediated T cell activation (A-D) by the Jurkat-NFAT-Luc reporter system, [Figure 5C] Detection of exemplary antibody TS-F2-4 and 5 mediated T cell activation (A-D) by the Jurkat-NFAT-Luc reporter system, [Figure 5D] Detection of exemplary antibody TS-F2-4 and 5 mediated T cell activation (A-D) by the Jurkat-NFAT-Luc reporter system, [Figure 5E] Shows detection of exemplary antibody TS-F2-4, 5 and 6 mediated T cell activation (E-F) by the Jurkat-NFAT-Luc reporter system. [Figure 5F]This shows the detection of exemplary antibody TS-F2-4, 5, and 6-mediated T cell activation (EF) using the Jurkat-NFAT-Luc reporting system. [Figure 6A] This shows the detection of exemplary antibody TS-F7-1 and 7-2-mediated T cell activation using the Jurkat-NFAT-Luc reporting system. [Figure 6B] This shows the detection of exemplary antibody TS-F7-1 and 7-2-mediated T cell activation using the Jurkat-NFAT-Luc reporting system. [Figure 6C] This shows the detection of exemplary antibody TS-F7-1 and 7-2-mediated T cell activation using the Jurkat-NFAT-Luc reporting system. [Figure 6D] This shows the detection of exemplary antibody TS-F7-1 and 7-2-mediated T cell activation using the Jurkat-NFAT-Luc reporting system. [Figure 7] This shows the detection of exemplary antibody TS-F7-3 and 7-4-mediated T cell activation using the Jurkat-NFAT-Luc reporting system. [Figure 8] This illustrates the activation of CD4+ T cells in an exemplary antibody-mediated PBMC. [Figure 9] This shows an example of CD8+ T cell activation in antibody-mediated PBMCs (B). [Figure 10] This demonstrates the exemplary antibody-mediated PBMC killing effect against H929 cells. [Figure 11A] This shows the cytokine release levels of exemplary antibody-induced PBMCs associated with the NCl-H929 cell killing process. [Figure 11B] This shows the cytokine release levels of exemplary antibody-induced PBMCs associated with the NCl-H929 cell killing process. [Figure 11C] This shows the cytokine release levels of exemplary antibody-induced PBMCs associated with the NCl-H929 cell killing process. [Figure 11D] This shows the cytokine release levels of exemplary antibody-induced PBMCs associated with the NCl-H929 cell killing process. [Figure 12]This demonstrates the tumor-suppressing effect of exemplary antibodies in a humanized mouse model of H929 tumor. [Figure 13] This shows the expression levels of GPRC5D and BCMA on the surface of different multiple myeloma cells. [Modes for carrying out the invention]

[0010] Unless otherwise specified, all technical and scientific terms used herein have the ordinary meanings understood by those skilled in the art. All publications, patent applications, patents, or other references referenced herein are incorporated in their entirety by citation. Furthermore, the materials, methods, and examples described herein are for illustrative purposes only and are not intended to limit them. Other features, purposes, and advantages of the present invention will become apparent from this specification, the drawings, and the appended claims.

[0011] I. Definition The term "approximately," when used with a number or figure, means covering a range of numbers or figures that is 5% smaller than the lower limit and 5% larger than the upper limit.

[0012] As used herein, the terms “contain” or “include” mean include the elements, integers or steps described, but do not exclude any other elements, integers or steps.

[0013] The term “antibody” is used herein in its broadest sense, meaning a protein containing an antigen-binding site, and includes, but is not limited to, natural and artificial antibodies of various structures, including 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 naturally occurring glycoproteins containing 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)) interposed between them by a conservative region (framework region (FR)). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The constant region does not directly participate in antibody-antigen binding, but it exhibits various effector functions.

[0015] The term "antigen-binding fragment" refers to a part or fragment of an intact or complete antibody with fewer amino acid residues than that of an intact or complete antibody, which can bind to an antigen or compete with the complete antibody (i.e., the intact antibody from which the antigen-binding fragment originates). 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 VL, VH, CL, and CH1 domains; for example, a Fab fragment can be obtained by digesting a complete antibody with papain. F(ab')2, produced by digesting a complete antibody below the disulfide bond in the hinge region using pepsin, is a dimer of Fab' and is a bivalent antibody fragment. F(ab')2 can be reduced by disrupting the disulfide bond in the hinge region under neutral conditions, thereby converting the F(ab')2 dimer to a Fab' monomer. The Fab' monomer is essentially a Fab fragment with a hinge region (for a detailed explanation of other antibody fragments, see Fundamental Immunology, WEPaul (ed.), Raven Press, NY (1993)). The Fv fragment consists of the single-arm VL and VH domains of the antibody. Although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be linked by a synthetic linkage peptide that can produce these two domains as a single protein chain using recombination, and the VL and VH regions pair up in this single protein chain to form a single-chain Fv. The antibody fragment can be obtained by chemical methods, DNA recombination, or protein enzymatic digestion.

[0016] As used herein, the terms “antigen-binding site” and “antigen-binding domain” are interchangeable and refer to the region of an antibody molecule that actually binds to an antigen. Preferably, the antigen-binding site of the antibody molecule used in the present invention includes a VH / VL pair consisting of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH), the VH / VL pair may be contained in a single polypeptide chain or in two isolated polypeptide chains. In a preferred embodiment, the antibody molecule of the present invention includes 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, or hexavalent 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 to a different epitope of different antigens. The antibodies provided herein are tripspecific antibodies against GPRC5D, BCMA, and CD3.

[0018] The term "immunoglobulin molecule" refers to a protein that has the structure of a naturally occurring antibody. For example, IgG-class immunoglobulins are heterotetrameric glycoproteins with approximately 150,000 daltons, consisting of two disulfide-linked light chains and two heavy chains. From the N-terminus to the C-terminus, each immunoglobulin heavy chain has one heavy chain variable region (VH), also called a 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 a 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 such as 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 based on the amino acid sequence of their constant domain. IgG immunoglobulins basically consist of two Fab molecules and two dimerized Fc regions linked via an immunoglobulin hinge region.

[0019] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain involved in antibody-antigen binding. The variable domains of the heavy and light chains of natural antibodies typically have similar structures, where each domain contains four conserved framework regions (FRs) and three complementarity-determining regions. In some situations, a single VH or VL domain can provide sufficient antigen-binding specificity.

[0020] The "complementarity-determining region," or "CDR region," or "CDR," or "hypervariable region," is a region in the antibody variable domain that is sequence-hypervariable and structurally formed and determined loop ("hypervariable loop") and / or contains antigen contact residues ("antigen contact sites"). CDRs primarily play a role in binding to antigen epitopes. Heavy chain and light chain CDRs are numbered sequentially from the N-terminus and are usually called CDR1, CDR2, and CDR3. CDRs located within the heavy chain variable domain of an antibody are also called HCDR1, HCDR2, and HCDR3, and CDRs located within the light chain variable domain of an antibody are called LCDR1, LCDR2, and LCDR3. The CDR sequence of a given light chain or heavy chain variable region can be determined using various methods known in the field, including Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop; Kabat based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USD Department of Health and Human Services, National Institutes of Health (1987)); AbM (University of Bath); Contact (University College London); the International ImmunoGeneTics database (IMGT) (International Immunogenetic Information System, World Wide Web imgt.cines.fr / ); and the North CDR definition based on affinity propagation clustering that utilizes 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, use different definition ranges for the CDR area numbered by Kabat and Chothia. [Table 1]

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

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

[0024] The terms “Fc domain” or “Fc region” are used herein to define the C-terminal region of an immunoglobulin heavy chain, including at least some constant regions. The terms include native sequence Fc regions and variant Fc regions. A native immunoglobulin “Fc domain” includes two or three constant domains, namely a CH2 domain, a CH3 domain, and a selectable CH4 domain. For example, in a native antibody, the immunoglobulin Fc domain includes second and third constant domains (CH2 and CH3 domains) derived from two heavy chains of IgG, IgA, and IgD antibodies, or second, third, and fourth constant domains (CH2, CH3, and CH4 domains) derived from two heavy chains of IgM and IgE 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 known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interests, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0025] The term "effector function" refers to the biological activity directly linked to the immunoglobulin Fc region, which varies depending on the immunoglobulin isotype. Examples of immunoglobulin effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), cytokine secretion, antigen uptake by immune complex-mediated 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) a constant region or part thereof is modified, substituted, or exchanged with a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug) that confers a new type, effector function, and / or species-specific properties to the constant region or chimeric antibody by altering, substituting, or exchanging its constant region or part thereof. For example, a mouse antibody can be modified by changing its constant region to a constant region derived from human immunoglobulin. Because it has been modified to a human constant region, the chimeric antibody retains its specificity for antigen recognition but 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) while exhibiting low immunogenicity 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 rest of the antibody to the corresponding human-derived portion (i.e., the non-binding portion in the constant and variable regions is the corresponding portion of the 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 technique disclosed in US5,766,886.

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

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

[0030] The "percentage of amino acid sequence identity (%)" refers to the percentage of amino acid residues in the candidate sequence that match the amino acid residues of the specific amino acid sequence shown herein, when a candidate sequence is compared with the specific amino acid sequence shown herein, and gaps are introduced as necessary to achieve the maximum possible percentage of sequence identity, and no conservative substitutions are considered part of the sequence identity. In some embodiments, the present invention considers variants of the antibody molecule of the present invention, which have a substantial degree of identity with the antibody molecule and its sequence specifically disclosed herein, for example, at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more. The variants may include conservative modifications. In the case of polypeptide sequences, "conservative modifications" include substitutions, deletions, or additions to the polypeptide sequence, where certain amino acids are replaced with chemically similar amino acids. Tables of conservative substitutions that provide functionally similar amino acids are well known in this field. Such conservatively modified variants are additive to the polymorphic variants, interspecific homologs, and alleles of the present invention, and they are not excluded. The following are eight pairs of amino acids that are conserved substitutions with each other. 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, for example, Creighton, Proteins (1984)). In some embodiments, the term “conservative sequence modification” refers to amino acid modifications that have no apparent effect on or do not alter the binding characteristics of an antibody containing an amino acid sequence.

[0031] The term "host cell" refers to a cell into which an exogenous polynucleotide has already been introduced, and includes the offspring of such cells. The host cell includes "transformed organisms" and "transformed cells," and "transformed organisms" and "transformed cells" include cells transformed from the primary generation and their derived offspring. The host cell may be any type of cell line used to produce the antibody molecule of the present invention, and includes eukaryotic cells such as mammalian cells, insect cells, and yeast cells, and prokaryotic cells such as E. coli cells. The host cell includes cultured cells, and includes cells inside genetically modified animals, genetically modified plants, or cultured plant or animal tissues.

[0032] The term "expression vector" refers to a vector containing recombinant polynucleotides and including an expression regulatory sequence effectively 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 present in an in vitro expression system. Expression vectors include all known in the art, including cosmids, plasmids (e.g., nude or liposome-containing), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, adeno-associated viruses) into which recombinant polynucleotides are incorporated.

[0033] Mammals include, but are not limited to, domesticated 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 is humans.

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

[0035] The terms “tumor” and “cancer” are used interchangeably herein and include solid tumors and humoral tumors.

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

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

[0038] The term "CD3" refers to the T cell engagement antigen, the glycoprotein CD3 on the surface of T cells (e.g., the human CD3 protein with accession number UniProt P07766). In this specification, "antigen binding specificity to CD3" refers to an antibody or antibody fragment that specifically binds to CD3, e.g., scFv or Fab. In one embodiment, the antigen-binding site of the antibody molecule of the present invention that binds to CD3, as detected by biofilm layer optical interferometry, may have high affinity binding activity to the CD3 epsilon chain, e.g., a KD value of 1 nM to 50 nM, e.g., 0.1 nM to 5 nM. Preferably, in the tripspecific antibody molecule of the present invention, a high affinity CD3 antigen-binding site having a KD value of less than 50 nM is used. In one embodiment, the antigen-binding specificity is cross-reactive to human and monkey CD3.

[0039] When describing the 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] In this specification, a “knob-in-hole” mutation means introducing mutations into a first Fc polypeptide and a second Fc polypeptide, respectively, by the “knob-in-hole” technique to form a projection (“knob”) and a complementary cavity (“hole”) at the interface of the first Fc polypeptide and the interface of the second Fc polypeptide. As is known in the art, the “knob-in-hole” technique can facilitate the precise binding of each strand of an antibody molecule by altering the interface between different strands of the antibody molecule. Generally, the technique relates to introducing a “projection” at the interface of one strand and introducing a “cavity” corresponding to the interface of the other strand that pairs with it, so that the projection can be positioned in the cavity. One preferred interface includes the CH3 domain of the heavy chain constant domain of one strand and the CH3 domain of the heavy chain constant domain of the other strand that pairs with it. A protrusion can be constructed by replacing the small amino acid side chain of the CH3 domain interface of the heavy chain constant domain derived from one chain with a relatively large side chain (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a relatively small side chain (e.g., alanine or threonine), a compensatory cavity of the same or similar size as the protrusion is constructed at the interface of the CH3 domain of the heavy chain constant domain of the other paired chain. Another selectable interface, as in the Fab fragment described above, includes the CL domain of the light chain and the CH1 domain of the heavy chain, and facilitates precise heterodimerization between the two chains of the Fab fragment by constructing a protrusion-cavity interaction.

[0041] "Single-stranded variable fragment" or "scFv" is used herein to refer to a single-stranded 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-stranded variable fragment" or "dsscFv" is used herein to refer to a single-stranded variable fragment stabilized by a disulfide bond, 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] In this specification, “Fv fragment” is used to refer to an antibody fragment containing a heavy chain variable domain VH and a light chain variable domain VL. In this specification, “disulfide bond-stabilized variable fragment” or “dsFv” is used to refer to an Fv fragment stabilized by a disulfide bond artificially introduced between the VH and VL domains.

[0043] In this specification, "single-domain antibody" or "sdAb" is used to refer to an antibody fragment consisting of a single variable antibody domain, such as VH or VL, e.g., a heavy-chain variable domain derived from a camelid heavy-chain antibody, or a VH-like single domain (v-NAR) derived 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 derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks).

[0044] In this specification, “Fab fragment” or “Fab” is used to refer to an immunoglobulin fragment comprising two polypeptide chains, an immunoglobulin heavy chain variable domain VH, a heavy chain constant domain CH1, a light chain variable domain VL, and a light chain constant domain CL, wherein one polypeptide chain comprises VH and one constant region selected from CH1 and CL from the N-terminus to the C-terminus, and the other polypeptide chain comprises VL and the other constant region selected from CL and CH1 from the N-terminus to the C-terminus, wherein the VH domain and the VL domain pair to form an antigen-binding site. In this specification, if one polypeptide chain of Fab contains VH linked to CL and the other polypeptide chain contains VL linked to CH1, then Fab is also called crossFab.

[0045] In this specification, “single-stranded Fab” or “scFab” is used to refer to a single-stranded polypeptide formed by linking two strands of Fab fragments with a linker.

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

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

[0048] In this specification, the terms “flexible linking peptide,” “linking peptide,” or “linker” refer to a short amino acid sequence consisting of amino acids, for example, a hinge region derived from glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or from an immunoglobulin. In one embodiment, the linking peptide has an amino acid length of 5 to 50 amino acids, for example, 10, 15, 20, 25, or 30 amino acids. In one embodiment, the linking peptide comprises an amino acid sequence (G4S)n, where n is an integer of 1 or more, for example, n is an integer of 2, 3, 4, 5, 6, or 7. In one embodiment, the linking peptide comprises an amino acid sequence TS(G4S)n, where n is an integer of 1 or more, for example, n is an integer of 2, 3, 4, 5, 6, or 7. In further embodiments, the linking peptide is a hinge region derived from immunoglobulin, for example, an amino acid sequence of a hinge region containing "CPPC", such as the amino acid sequence "EPKSCDKTHTCPPCP" (SEQ ID NO: 114) or "EPKSSDKTHTCPPCP" (SEQ ID NO: 115). Linking peptides that link each domain of an antibody molecule applicable to the present invention may be, but are not limited to, 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), LRQRDGERP (SEQ ID NO: 123), LRQKDGGGSERP (SEQ ID NO: 124), and GTSTGSGKPGSGEGSTKG (SEQ ID NO: 125). Alternatively, the three-dimensional structure of proteins and peptides can be simulated using computer programs, or suitable flexible linked peptides can be rationally designed using phage display methods.

[0049] I. The Trispecific Antibody Molecules of the Present Invention The triplicate antibody molecule of the present invention is a Y-type antibody molecule. In this specification, the term "Y-type antibody molecule" refers to an antibody molecule having a Y-type structure comprising two antibody arms and one stem portion, where the two antibody arms are linked to the stem portion of the Y-type structure, which is formed by two branches of the Y-type structure and paired by two antibody Fc domains. One typical Y-type antibody molecule is the immunoglobulin IgG molecule. The Y-type structure of the immunoglobulin IgG molecule consists of three regions: two antibody arms (Fab) and one stem portion (Fc), where a flexible hinge region (Hinge) connects the antibody stem portion (Fc) to the antibody arms (Fab). In the IgG molecule, the two arms are used for specific binding of the antigen, while the stem portion determines the antibody type and functional characteristics. In this specification, the term Y-type antibody molecule also covers molecules having the same or similar Y-type structure as the IgG molecule, such as Y-type molecules having complex components in the antibody arms and / or stem of the antibody. Preferably, the antibody molecule of the present invention is a triple-specific antibody molecule having a complex component.

[0050] In the Y-type antibody molecule of the present invention, the antibody arms may consist of antigen-binding domains such as Fab, scFv, and sdAB, and the antigen-binding domains constituting the two antibody arms may target homologous or heterologous antigens and / or epitopes and have homologous or heterologous binding specificity. In the Y-type antibody molecule of the present invention, the stem of the antibody molecule may consist of two paired immunoglobulin Fc domains, where each of 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 of the stem contain mutations that promote their pairing and dimerization, such as complementary "knob-in-hole" mutations. Furthermore, the Fc domain of the stem may also contain other mutations, such as cysteine ​​mutations (which promote the formation of a disulfide bond 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 L234A / L235A mutation combination). The two antibody arms of the Y-type antibody molecule of the present invention may be covalently bonded to each other's Fc domains of the stem either directly or via linking peptides, preferably the arms are linked to the Fc domains of the stem via the hinge region of immunoglobulin.

[0051] In addition to the arms and stem, the Y-type antibody molecule of the present invention preferably further comprises a complex component compounded with the arms and / or stem, the complex component comprising additional antigen-binding domains such as Fab, scFv, and sdAB, preferably scFv. The antigen-binding domain in the complex component can confer different antigen-binding specificities from those of the antibody arms. For example, the two antibody arms of the Y-type molecule may each contain antigen-binding domains that provide first and second antigen-binding specificities, respectively, and the complex component of the Y-type molecule may contain an antigen-binding domain that provides a third antigen-binding specificity, and the first, second, and third antigen-binding specificities are each different. Alternatively, the two antibody arms of the Y-type molecule may each contain an antigen-binding domain that provides first antigen-binding specificity, and the complex component of the Y-type molecule may contain antigen-binding domains that provide second and third antigen-binding specificities, and the first, second, and third antigen-binding specificities are each different.

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

[0053] Depending on the structure of the arms, stem, and complex components, the Y-type antibody molecule of the present invention may contain at least two polypeptide chains, for example, two or four polypeptide chains. In this specification, polypeptide chains containing a stem Fc domain are referred to as heavy chains, while polypeptide chains not containing an Fc domain are referred to as light chains. Thus, in some embodiments, the Y-type molecule of the present invention is a double-chain molecule containing a first heavy-chain polypeptide chain and a second heavy-chain polypeptide chain, and in some other embodiments, the Y-type molecule of the present invention is a quadruple-chain molecule containing two heavy-chain polypeptide chains and two light-chain polypeptide chains. In the Y-type molecule of the present invention containing a complex component, the complex component may be compounded with a heavy-chain polypeptide chain of the antibody molecule (e.g., the C-terminus of the Fc domain of the stem portion) or with a light-chain polypeptide chain of the antibody molecule (e.g., the C-terminus of the Fab light chain of the antibody arm containing the Fab antigen binding site). Therefore, for example, the antibody of the present invention may include two heavy polypeptide chains and two light polypeptide chains, where the first heavy chain and the first light chain pair to form one antibody arm of the antibody, the second heavy chain and the second light chain pair to form another antibody arm of the antibody, and the Fc domain of the first heavy chain and the Fc domain of the second heavy chain pair to form an antibody stem, where the C-terminus of the first or second heavy chain further includes a single-chain antibody fragment (such as scFv) that forms an antibody complex component, or the C-terminus of the first and second light chains further includes a single-chain antibody fragment (such as scFv) that forms an antibody complex component.

[0054] In one embodiment, the present invention therefore provides a triplicate Y-type antibody molecule. The Y-type antibody molecule of the present invention has the following formula: (M1:M2-(X1)p)-Fc::Fc-(X2)q (Formula I) It has a structure, and in the formula, M1 and M2 represent the first and second antibody arms of a Y-type antibody molecule containing antigen-binding sites, respectively. Here, the antigen-binding sites of the first and second antibody arms may be homologous or different. Fc::Fc represents the stem of a Y-type antibody molecule located at the C-terminus of the first and second antibody arms, and consists of a paired and dimerized first Fc domain and a second Fc domain. X1 represents a complex component compounded at the C-terminus of the antibody arm, and X2 represents a complex component compounded at the C-terminus of the stem, where the complex component includes an antigen-binding site, where the complex component is compounded to the antibody molecule directly or via a linked peptide, and In the formula, p and q represent integers 0, 1, or 2, respectively, and p and q are not both 0 at the same time. Here, the antibody is specifically bound to GPRC5D, BCMA, and CD3 via the antibody arm and complex components. Preferably, when the first antibody arm and the second antibody arm bind to different antigens, either p or q is 0, where antibody arms M1 and M2 provide first and second antigen-binding specificity, and the complex component provides third antigen-binding specificity, or Preferably, when the first antibody arm and the second antibody arm bind to the same antigen, neither p nor q is 0, where antibody arms M1 and M2 provide first antigen-binding specificity, and complex components X1 and X2 provide second and third antigen-binding specificity, respectively.

[0055] In some embodiments, the antibody arms (M1 and M2) of the antibody molecule include antigen-binding sites selected from Fab or scFab (preferably Fab), and either p or q is 0, where antibody arms M1 and M2 are bound to first and second antigens, respectively, and a complex component (X1 or X2) is bound to a third antigen, where the first, second, and third antigens are distinct from each other and independently selected from GPRC5D, BCMA, and CD3. Preferably, when p=0, q=1, and the complex component X2 is compounded to one of the two Fc domains of the stem of the antibody molecule, or Preferably, when q=0, p=2, and the complex component X1 is compounded into both the Fab light chains M1 and M2 of the antibody arm of the antibody molecule.

[0056] In some embodiments, the antibody arms (M1 and M2) of the antibody molecule include antigen-binding sites selected from Fab or scFab (preferably Fab), where neither p nor q is 0, and antibody arms M1 and M2 are bound to the same first antigen, and complex components X1 and X2 are bound to the second and third antigens, respectively, where the first, second, and third antigens are distinct from each other and independently selected from GPRC5D, BCMA, and CD3. Preferably, p=2 and q=2, where complex component X1 is compounded to both Fab light chains M1 and M2 of the antibody arm of the antibody molecule, and complex component X2 is compounded 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 include antigen-binding sites selected from scFv (preferably dsscFv), with p=0 and q=1 or 2, where antibody arms M1 and M2 are bound to the first and second antigens, respectively, and complex component X2 is bound to the third antigen, where the first, second, and third antigens are distinct from each other and independently selected from GPRC5D, BCMA, and CD3. Preferably, when p=0, q=1, and the complex component X2 is compounded to the C-terminus of one of the two Fc domains of the stem of the antibody molecule.

[0057] In any one of the above embodiments, the complex component preferably includes an antigen-binding site selected from scFv, dsFv, and dsAb, preferably scFv, more preferably dsscFv. In any one of the embodiments described above, preferably, the first Fc domain and the second Fc domain include a hinge region having a "CPPC" amino acid residue, thereby facilitating the correct pairing of polypeptide chains in the antibody molecule of the present invention by forming an interchain disulfide bond between the first and second Fc domains. Preferably, in the antibody molecule of the present 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 embodiments described above, the complex component is preferably compounded to the stem and / or arms of the antibody molecule by a flexible linking peptide. Preferably, the length of the linking peptide is 5 to 15 amino acids, for example, 10, 12, or 15 amino acids. Preferably, the linking peptide comprises the amino acid sequence TS(G4S)n or (G4S)n, where n=1, 2, or 3, preferably n=2.

[0058] Therefore, in some preferred embodiments, when either p or q in formula I is 0, the present invention is expressed in the following formula: (M1:M2-(X1)p)-Fc::Fc-(X2)q (Formula I) We provide an IgG-like antibody molecule having the structure of the formula, M1 and M2 each contain Fab or scFab, preferably Fab, that binds to the first and second antigens, respectively. In the formula, if p=0, X2 contains scFv that binds to the third antigen, or If q=0, X1 contains scFv that binds to the third antigen. Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3. In some further preferred embodiments, when p=0 in formula I, the present invention relates to the following formula: (M1:M2)-Fc::Fc-(X2)q, (Format_2) We provide an IgG-like antibody molecule having the structure of the formula, q=1, M1 and M2 each contain Fab or scFab, preferably Fab, that binds to the first and second antigens, and X2 contains scFv that binds to the third antigen, Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3.

[0059] In some further preferred embodiments, when q=0 in formula I, the present invention relates to the following formula: (M1:M2-(X1)p)-Fc::Fc, (Format_7) We provide an IgG-like antibody molecule having the structure of the formula, p=2, M1 and M2 each contain Fabs that bind to the first and second antigens, and X2 contains scFv that binds to the third antigen, Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3.

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

[0061] In some further embodiments, when p=0 and q=1 in equation I, the present invention relates to the following equation: (M1:M2)-Fc::Fc-X2, (Format_1) We provide an IgG-like antibody molecule having the structure of the formula, M1, M2, and X2 each contain scFv that bind to the first, second, and third antigens, respectively. Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3.

[0062] The antibody arms, complex components, and stems constituting the triplicate antibody molecule of the present invention will be described in further detail below. As will be apparent to those skilled in the art, these descriptions apply to any triplicate antibody molecule according to the present invention, for example, the molecule of formula I, and molecules having structures of Formats 1, 2, 6, and 7.

[0063] Antibody arms and complex components of the antibody molecule of the present invention The antibody molecule of the present invention provides antigen-binding sites that specifically bind to first, second, and third antigens, through the antibody arm and antigen-binding sites of the complex component, where the first, second, and third antigens are distinct from each other and independently selected from tumor-associated antigen GPRC5D, tumor-associated antigen BCMA, and T-cell engagement antigen CD3.

[0064] In one embodiment, the present 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 first and second antigen-binding sites, respectively, and the complex component provides a third antigen-binding site.

[0065] In other embodiments, the present invention provides an antibody molecule of formula I, where, if neither p nor q is 0, the first and second antibody arms of the antibody provide a first antigen-binding site, a complex component compounded at the C-terminus of the antibody arm provides a second antigen-binding site, and a complex component compounded at the antibody stem provides a third antigen-binding site.

[0066] The antigen-binding sites of the tripspecific antibody molecule applicable to the present invention (including antigen-binding sites that specifically bind to GPRC5D, BCMA, and CD3) may include 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 sites in the antibody arm and the complex component may independently be scFv, dsFv, Fab, scFab, or sdAb. Preferably, in some embodiments, the antigen-binding sites of the antibody arm are selected from Fab and scFab, preferably Fab. Preferably, in some other embodiments, the antigen-binding sites of the complex component are selected from scFv, dsFv, and dsAb, preferably scFv, more preferably scFv with a stable disulfide bond (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 complex component is scFv, particularly dsscFv.

[0067] In this specification, Fab_GPRC5D, Fab_BCMA, and Fab_CD3 are used to represent Fab-type antigen-binding sites that bind to GPRC5D, BCMA, and CD3, respectively. In this specification, ScFv_GPRC5D, ScFv_BCMA, and ScFv_CD3 are used to represent scFv-type antigen-binding sites that bind to GPRC5D, BCMA, and CD3, respectively.

[0068] In some embodiments, the Fab antigen-binding site in the antibody molecule of the present invention is composed of two polypeptide chains containing immunoglobulin VH, CH1, VL, and CL domains, where VH and VL are paired and CH1 and CL are paired to form the antigen-binding site. In some embodiments, in Fab, one chain contains VH and CH1 (i.e., VH-CH1) from the N-terminus to the C-terminus, and the other chain contains VL and CL (i.e., VL-CL) from the N-terminus to the C-terminus. In some other embodiments, in Fab, one chain contains VH and CL (i.e., VH-CL) from the N-terminus to the C-terminus, and the other chain contains VL and CH1 (i.e., VL-CH1) from the N-terminus to the C-terminus. In some embodiments, Fab constitutes the antigen-binding site of the antibody arm of the antibody molecule of the present invention. In the embodiments described above, the Fab can be fused to the N-terminus of the Fc domain of the antibody stem by the C-terminus of the chain containing VH, or, wherein this embodiment, the Fab is fused to the N-terminus of the Fc domain of the antibody stem by the C-terminus of the chain containing VL. Preferably, the Fab comprises a VH-CH1 chain and a VL-CL chain, and is bound to the Fc region of the antibody stem by the C-terminus of the VH-CH1 chain. Preferably, if the antibody molecule of the present invention includes a complex component compounded in an antibody arm, the complex component is compounded to the C-terminus of a Fab chain not linked to the antibody stem. In this specification, a Fab chain not linked to an antibody stem is also called a Fab light chain. Thus, in some embodiments, the antibody molecule of the present invention has a complex component compounded 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 present invention consists of a single polypeptide chain comprising immunoglobulin VH, CH1, VL, and CL domains, where VH and VL are paired and CH1 and CL are paired to form the antigen-binding site. In some embodiments, the scFab comprises VH-CH1, a linker, and VL-CL from the N-terminus to the C-terminus. In some other embodiments, the scFab comprises VL-CL, a linker, and VH-CH1 from the N-terminus to the C-terminus. In some further embodiments, the scFab comprises VH-CL, a linker, and VL-CH1 from the N-terminus to the C-terminus. In some other embodiments, the scFab comprises VL-CH1, a linker, and VH-CL from the N-terminus to the C-terminus. In some embodiments, the scFab constitutes the antigen-binding site of the antibody arm of the antibody molecule of the present invention. In some embodiments, the scFab is fused to the N-terminus of the Fc domain of the antibody stem by the C-terminus of its polypeptide chain. Preferably, the scFab comprises VL-CL, a linker, and VH-CH1 from the N-terminus to the C-terminus, and the C-terminus of the CH1 domain binds to the Fc region of the antibody stem. Preferably, if the antibody molecule of the present invention includes an scFab antibody arm, the complex component of the antibody molecule is complexed only at the C-terminus of the Fc domain of the antibody stem.

[0070] In some embodiments, the Fab or scFab contained in the antibody molecule of the present invention includes a CH1 domain derived from IgG immunoglobulin, for example, the CH1 domain of IgG1, preferably the CH1 domain of human IgG1. In some preferred embodiments, the CH1 domain includes 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 contained in the antibody molecule of the present invention includes a kappa light chain constant domain derived from IgG immunoglobulin, for example, the CL of IgG1. κ Domain, preferably human IgG1 CL κThe domain is included. For example, the Fab or scFab includes a kappa light chain constant domain having the 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 included in the antibody molecule of the present invention is a lambda light chain constant region derived from IgG immunoglobulin, for example, CL of IgG1. λ Domain, preferably human IgG1 CL λ The domain is included. For example, the Fab includes a lambda light chain constant domain having the 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, if the antibody arms M1 and M2 of the antibody molecule of the present invention include Fab or scFab (preferably Fab) that bind to different antigens, the light chain constant domains of the Fab or scFab of antibody arm M1 and antibody arm M2 are different from each other. For example, in some embodiments, the Fab of antibody arm M1 includes a kappa light chain constant domain and the Fab of antibody arm M2 includes a lambda light chain variable domain, or the Fab of antibody arm M1 includes a lambda light chain constant domain and the Fab of antibody arm M2 includes a kappa light chain variable domain.

[0071] In some embodiments, the scFv antigen-binding site in the antibody molecule of the present invention is composed of a single polypeptide chain containing immunoglobulin VH and VL domains, where VH and VL are linked and paired by a linker to form an antigen-binding site. In some embodiments, the scFv is trans-constituent and includes VH, a linker, and VL (VH-linker-VL) from the N-terminus to the C-terminus. In some other embodiments, the scFv is cis-constituent and includes VL, a linker, and VH (VH-linker-VL) from the N-terminus to the C-terminus. 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 has an amino acid length of 10 to 50 amino acids, for example, 5 to 30 amino acids, for example, 15 amino acids or 20 amino acids. In one embodiment, the linker includes an 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, the scFv constitutes the antigen-binding site of the antibody arm of the antibody molecule of the present invention, and is fused to the N-terminus of the Fc domain of the antibody stem by the C-terminus of its polypeptide chain.

[0072] In some other embodiments, the scFv constitutes the antigen-binding site of the complex component of the antibody molecule of the present invention. In some embodiments, the scFv is fused to the C-terminus of the antibody stem and / or the C-terminus of the antibody arm by the N-terminus of its polypeptide chain. Preferably, in the antibody molecule of the present invention, the antibody arm comprises Fab, and the complex component comprises scFv, where the scFv is fused to the C-terminus of the antibody stem Fc and / or the C-terminus of the antibody arm Fab light chain by the N-terminus of its polypeptide chain. In some preferred embodiments, the scFv antigen-binding site in the antibody molecule of the present invention is a stable scFv with a disulfide bond, i.e., dsscFv. When the antigen-binding site is dsscFv, the following residue pair (the positions below are determined by Kabat numbers) is introduced between the VH and VL variable domains that mutate to scFv: It can be located between the 37th residue of VH + the 95th residue of VL, the 44th residue of VH + the 100th residue of VL, the 44th residue of VH + the 105th residue of VL, the 45th residue of VH + the 87th residue of VL, the 55th residue of VH + the 101st residue of VL, the 100th residue of VH + the 50th residue of VL, the 100b residue of VH + the 49th residue of VL, the 98th residue of VH + the 46th residue of VL, the 105th residue of VH + the 43rd residue of VL, and the 106th residue of VH + the 57th residue of VL. Preferably, in order to form dsscFv, the 44th residue of VH and the 100th residue of VL are introduced into the cysteine ​​substitution so that a disulfide bond can be formed between the two residues when VH and VL pair up.

[0073] antigen-binding site that binds to GPRC5D The GPRC5D antigen-binding site of an 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 included in the antibody arm or complex component may independently be scFv, dsFv, Fab, scFab, or sdAb, and preferably an antigen-binding site having the scFv or Fab structure described above. 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 complex component is selected from scFv, dsFv, and dsAb, preferably scFv, more preferably scFv with a stable disulfide bond (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; in another more preferred embodiment, the antibody comprises a complex component that binds to GPRC5D, and the GPRC5D antigen-binding site of the complex component is scFv, particularly dsscFv.

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

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

[0076] Alternatively, the antigen-binding site that binds to GPRC5D, for example, scFv or Fab, comprises one variant of the combination of CDR sequences, wherein the variant comprises at least one and 5, 4, 3, 2 or 1 or fewer amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in total across 1, 2, 3, 4, 5 or preferably 6 CDR regions, and preferably the heavy chain CDR3 remains unchanged.

[0077] In some preferred embodiments, the antigen-binding site that binds to GPRC5D, for example, the GPRC5D antigen-binding site having the scFv or Fab structure described above, is as follows: (a) A heavy chain variable domain comprising the amino acid sequence shown 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 the amino acid sequence shown 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 the amino acid sequence shown 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 the amino acid sequence shown 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) Heavy chain variable domains comprising the amino acid sequence shown in Sequence 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 VH selected from the amino acid sequence shown in SEQ ID NO: 100 or a heavy chain variable domain having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0078] In some preferred embodiments, the antigen-binding site that binds to GPRC5D, for example, the GPRC5D antigen-binding site having the scFv or Fab structure described above, is as follows: (a) A light chain variable domain comprising the amino acid sequence shown 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) Light chain variable domains comprising the amino acid sequence shown 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) Light chain variable domains comprising the amino acid sequence shown 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) Light chain variable domains comprising the amino acid sequence shown 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) Light chain variable domains comprising the amino acid sequence shown in Sequence 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 VL selected from the amino acid sequence shown in Sequence ID No. 101 or a light chain variable domain having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0079] In some preferred embodiments, the antigen-binding site that binds to GPRC5D, for example, the GPRC5D antigen-binding site having the scFv or Fab structure described above, is as follows: (a) A heavy chain variable domain comprising the amino acid sequence shown 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 shown 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 the amino acid sequence shown 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 shown 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 containing the amino acid sequence shown 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 containing the amino acid sequence shown 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 the amino acid sequence shown 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 shown 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 the amino acid sequence shown 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 the amino acid sequence shown 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 the amino acid sequence shown 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 having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0080] In some preferred embodiments, the antigen-binding site that binds to GPRC5D, for example, the GPRC5D antigen-binding site having the scFv or Fab structure described above, is as follows: (i) A heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 25, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 26, or (ii) A heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 27, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 28, or (iii) A heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 29, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 30, or (iv) A heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 31, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 32, (v) A heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 98, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 99, (vi) A combination of VH and VL sequences selected from a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 100 and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 101. In some preferred embodiments, if the antigen-binding site that binds to GPRC5D is a stable scFv with a disulfide bond, the scFv further includes 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 an 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 included in the antibody arm or complex component may independently be scFv, dsFv, Fab, scFab, or sdAb, and 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 complex component is selected from scFv, dsFv, and dsAb, preferably scFv, more preferably scFv with a stable disulfide bond (i.e., dsscFv). In one more preferred embodiment, the antibody includes an antibody arm that binds to BCMA, and the BCMA antigen-binding site of the antibody arm is Fab, and in another more preferred embodiment, the antibody includes a complex component that binds to BCMA, and the BCMA antigen-binding site of the complex component is scFv, particularly dsscFv.

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

[0083] In some preferred embodiments, the antigen-binding site that binds to BCMA, for example, the BCMA antigen-binding site having the scFv or Fab structure described above, is as follows: -HCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 33, -HCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 34, -HCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 35, -LCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 36, -LCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 37, -Contains the amino acid sequence shown in Sequence ID No. 38, or a combination of CDR sequences called LCDR3 consisting of the same, Alternatively, the antigen-binding site that binds to BCMA includes one variant of the combination of CDR sequences, wherein the variant includes at least one amino acid mutation (preferably an amino acid substitution, preferably a conservative substitution) in total across 1, 2, 3, 4, 5, or preferably 6 CDR regions, and preferably the heavy chain CDR3 remains unchanged.

[0084] In some preferred embodiments, the antigen-binding site that binds to BCMA, for example, the BCMA antigen-binding site having the scFv or Fab structure described above, includes a heavy chain variable domain VH having the amino acid sequence shown 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, the BCMA antigen-binding site having the scFv or Fab structure described above, includes a light chain variable domain VL having the amino acid sequence shown 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, the antigen-binding site that binds to BCMA, for example, the BCMA antigen-binding site having the scFv or Fab structure described above, includes a heavy chain variable domain VH having the amino acid sequence shown in SEQ ID NO: 39 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto, and includes a light chain variable domain VL having the amino acid sequence shown in SEQ ID NO: 40 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0087] Preferably, the antigen-binding site that binds to BCMA, for example, the BCMA antigen-binding site having the scFv or Fab structure described above, includes a VH domain having the amino acid sequence shown in SEQ ID NO: 39 and a VL domain having the amino acid sequence shown in SEQ ID NO: 40. In some preferred embodiments, if the antigen-binding site that binds to BCMA is an scFv with a stable disulfide bond, the scFv further includes a cysteine ​​substitution at position 44 of the VH domain and a cysteine ​​substitution at position 100 of the VL domain.

[0088] Antigen binding site that binds to CD3 The CD3 antigen-binding site of an 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 included in the antibody arm or complex component may independently be scFv, dsFv, Fab, scFab, or sdAb, and 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 complex component is selected from scFv, dsFv, and dsAb, preferably scFv, more preferably scFv with a stable disulfide bond (i.e., dsscFv). In one more preferred embodiment, the antibody includes an antibody arm that binds to CD3, and the CD3 antigen-binding site of the antibody arm is Fab; in another more preferred embodiment, the antibody includes a complex component that binds to CD3, and the CD3 antigen-binding site of the complex component is scFv, particularly dsscFv.

[0089] In some preferred embodiments, the antigen-binding site that binds to CD3, for example, the CD3 antigen-binding site having the scFv or Fab structure described above, includes the sequences of HCDR1, 2 and 3 of the heavy chain variable domain shown in SEQ ID NO: 48, and the sequences of LCDR1, 2 and 3 of the light chain variable domain shown in SEQ ID NO: 49. In some other preferred embodiments, the antigen-binding site that binds to CD3, for example, the CD3 antigen-binding site having the scFv or Fab structure described above, includes the sequences of HCDR1, 2 and 3 of the heavy chain variable domain shown in SEQ ID NO: 50, and the sequences of LCDR1, 2 and 3 of the light chain variable domain shown in SEQ ID NO: 49.

[0090] In some preferred embodiments, the antigen-binding site that binds to CD3, for example, the CD3 antigen-binding site having the scFv or Fab structure described above, is as follows: -HCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 41, -HCDR2 containing or consisting of the amino acid sequence shown in SEQ ID NO: 42 or 47, -HCDR3 containing or consisting of the amino acid sequence shown in Sequence ID No. 43, -LCDR1 containing or consisting of the amino acid sequence shown in Sequence ID No. 44, -LCDR2 containing or consisting of the amino acid sequence shown in Sequence ID No. 45, -Contains the amino acid sequence shown in Sequence ID No. 46, or a combination of CDR sequences called LCDR3 consisting of the same, Alternatively, the antigen-binding site that binds to CD3 includes one variant of the combination of CDR sequences, wherein the variant includes at least one and 5, 4, 3, 2 or 1 or fewer amino acid mutations (preferably amino acid substitutions, preferably conservative substitutions) in total across 1, 2, 3, 4, 5 or preferably 6 CDR regions, and preferably the heavy chain CDR3 remains unchanged.

[0091] In some preferred embodiments, the antigen-binding site that binds to CD3, for example, the CD3 antigen-binding site having the scFv or Fab structure described above, includes a heavy chain variable domain VH having the amino acid sequence shown 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, for example, the CD3 antigen-binding site having the scFv or Fab structure described above, includes a light chain variable domain VL having the amino acid sequence shown 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, for example, the CD3 antigen-binding site having the scFv or Fab structure described above, includes a heavy chain variable domain VH having the amino acid sequence shown 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, and includes a light chain variable domain VL having the amino acid sequence shown in SEQ ID NO: 49 or an amino acid sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0093] Preferably, the antigen-binding site that binds to CD3, for example, the scFv or Fab structure CD3 antigen-binding site described above, includes 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, for example, the scFv or Fab structure CD3 antigen-binding site described above, includes 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, if the antigen-binding site that binds to CD3 is an scFv with a stable disulfide bond, the scFv further includes a cysteine ​​substitution at position 44 of the VH domain and a cysteine ​​substitution at position 100 of the VL domain.

[0094] Stem of antibody molecule of the present invention The antibody molecule of the present invention includes a stem located at the C-terminus of the antibody arm, formed by a first Fc domain and a second Fc domain. In some embodiments, the first and second Fc domains are identical. In some 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 to which the present invention applies 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 a selectable CH4 domain. Preferably, the Fc domain of the antibody of the present invention includes CH2-CH3 from the N-terminus to the C-terminus, and more preferably includes a hinge region-CH2-CH3 from the N-terminus to the C-terminus. In the antibody molecule of the present invention, the Fc domain may include second and third constant domains (CH2 domain and CH3 domain) derived from IgG, IgA, and IgD antibodies, or it may include second, third, and fourth constant domains (CH2 domain, CH3 domain and CH4 domain) derived from IgM and IgE antibodies. In some embodiments, the Fc domain of the antibody molecule is an Fc domain from IgG, for example, an Fc domain from IgG1, IgG2, or IgG4, preferably an Fc domain from human IgG1.

[0096] As those skilled in the art will understand, depending on the expected use of the antibody molecule of the present invention, the antibody molecule of the present invention may include modifications that alter the effector function of the Fc domain. 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 modification of the glycosylation of the Fc domain, use of an Fc isotype having naturally reduced or eliminated effector functions, and modification of the amino acid sequence of the Fc domain.

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

[0098] In another embodiment, the effector function is reduced or eliminated by modifying the amino acid sequence of at least one Fc domain. The Fc domain modification involves introducing 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 the binding of the Fc domain to one or more Fc receptors, or introducing a point mutation at a position selected from 270, 322, 329, and 321 that impairs C1q binding.

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

[0100] As those skilled in the art will understand, if the antibody molecule of the present invention is an asymmetric antibody molecule (e.g., an antibody molecule of Format_2 and Format_7), then in order to facilitate the 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, Knob-in-Hole technology can be used to introduce complementary Knob and Hole mutations into the first and second Fc domains.

[0101] Accordingly, in some embodiments, the present invention provides a trispecific antibody molecule in which the first Fc domain and the second Fc domain each contain first and second heterodimerization mutations 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 includes a Knob mutation and the second heterodimerization mutation of the second Fc domain includes a Hole mutation complementary to the Knob mutation, or the first heterodimerization mutation of the first Fc domain includes a Hole mutation and the second heterodimerization mutation of the second Fc domain includes a Knob mutation complementary to the 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 is i) In some cases, the homodimer Fc-region of a human IgG1 subclass having mutations L234A and L235A, or ii) Depending on the case, the Fc-region of a human IgG4 subclass homodimer having mutations P329G, S228P and L235E, or iii) A heterodimer Fc-region, of which a) One Fc-region polypeptide contains mutation T366W, another Fc-region polypeptide contains mutations T366S, L368A and Y407V, or b) One Fc-region polypeptide contains mutations T366W and Y349C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and S354C, or c) One Fc-region polypeptide contains mutants T366W and S354C, and another Fc-region polypeptide contains mutants T366S, L368A, Y407V and Y349C, heterodimer Fc-region, or iv) A heterodimer Fc-region of a human IgG1 subclass, wherein both of the two Fc-region polypeptides contain mutants L234A and L235A, a) One Fc-region polypeptide contains mutation T366W, another Fc-region polypeptide contains mutations T366S, L368A and Y407V, or b) One Fc-region polypeptide contains mutations T366W and Y349C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and S354C, or c) A heterodimer Fc region of a human IgG1 subclass, in which one Fc-region polypeptide contains mutations T366W and S354C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and Y349C. or v) A heterodimer Fc-region of a human IgG4 subclass, wherein both of the Fc-region polypeptides contain mutants P329G, S228P, and L235E, a) One Fc-region polypeptide contains mutation T366W, another Fc-region polypeptide contains mutations T366S, L368A and Y407V, or b) One Fc-region polypeptide contains mutations T366W and Y349C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and S354C, or c) A heterodimer Fc region of a human IgG4 subclass, in which one Fc-region polypeptide contains mutations T366W and S354C, and another Fc-region polypeptide contains mutations T366S, L368A, Y407V and Y349C. This provides a triplicate antibody molecule containing the following:

[0103] In some embodiments, the Fc domain of the antibody molecule of the present invention may further contain other mutations that facilitate the purification of heteropolymer antibodies. For example, the H435R mutation may be introduced into one of the first and second Fc domains (e.g., the Fc domain having the Hole mutation) to facilitate the purification of targeted heteropolymer antibodies 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 to which the present invention applies, 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 include an amino acid sequence of a hinge region derived from IgG1, or an amino acid sequence selected from (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n is at least an integer of 1. Preferably, the antibody stem can 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 heteropolymer antibody of the present invention including a hinge region, a mutation, such as C220S, can be introduced into the hinge region to promote the formation of a target heteropolymer antibody.

[0105] Exemplary triple-specific antibody molecules Format_2 In some embodiments, when p=0 and q=1, the antibody molecule of the present invention has a structure of (M1:M2)-Fc::Fc-(X2)q. Thus, in the antibody molecule, there is only a complex component X2 compounded to the stem. In the antibody molecule, preferably, the antibody arms M1 and M2 include Fab antigen-binding sites that bind to a first antigen and a second antigen, respectively, and the complex component X2 includes 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] Therefore, in some preferred embodiments, the present invention provides a triplicate Y-type antibody molecule, the antibody molecule being as follows: (M1:M2)-Fc::Fc-(X2)q, (Format_2) It has the structure of the form, where q=1, M1 and M2 represent the first and second antibody arms of the antibody molecule, respectively, and M1 and M2 each contain Fab or scFab, preferably Fab, that binds to the first and second antigens. Fc::Fc represents the stem of the antibody molecule, consisting of a paired and dimerized first Fc domain and a second Fc domain, where the first and second antibody arms are ligated directly or via a linking peptide (preferably a hinge region) to the N-terminuses of the first and second Fc domains, respectively. X2 represents a complex component compounded at the C-terminus of the stem, where the complex component includes an scFv that binds to the third antigen, where X2 is compounded at the C-terminus of the first Fc domain directly or via a linked peptide, or at the C-terminus of the second Fc domain. Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3.

[0107] In some embodiments, the present invention provides a triplicate antibody molecule having a Format_2 structure, where, (a): The antibody arms M1 and M2 of the molecule contain Fab that binds to GPRC5D and Fab that binds to CD3, respectively, and the complex component X2 contains scFv that binds to BCMA, and the molecule has the structure (Fab_GPRC5D:Fab_CD3)-Fc::Fc-(scFv_BCMA) (for example, molecules TS-F2-1 and 3-6 of the example), or (b): The antibody arms M1 and M2 of the molecule contain Fab that binds to BCMA and Fab that binds to CD3, respectively, and the complex component X2 contains scFv that binds to GPRC5D, and the molecule has a structure of (Fab_BCMA:Fab_CD3)-Fc::Fc-(scFv_GPRC5D) (for example, the TS-F2-2 molecule of the example), or (c): The antibody arms M1 and M2 of the molecule each contain Fab that binds to GPRC5D and Fab that binds to BCMA, respectively, and the complex component X2 contains scFv that binds to 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, the GPRC5D antigen-binding site of the present invention having the scFv or Fab structure described above, and in particular, the 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 present invention described above, the Fab or scFv that binds to BCMA may be any suitable Fab or scFv that specifically binds to BCMA, for example, the BCMA antigen-binding site of the present invention having the scFv or Fab structure described above, and in particular, the scFv or Fab having the VH and VL amino acid sequences described above.

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

[0111] In any embodiment of the Format_2 antibody molecule of the present invention described above, the antibody molecule may include any antibody stem structure of the present invention applicable to Format_2 as described above. For example, in the Format_2 asymmetric trispecific antibody molecule of the present invention, mutations that promote heterodimerization of the first and second Fc domains, particularly complementary "knob-in-hole" mutations, can be introduced into the first and second Fc domains of the stem, preferably to facilitate the correct pairing of the antibody molecule polypeptide chains. For example, a knob mutation can be introduced into the first Fc domain and a complementary hole mutation into the second Fc domain, or vice versa. In this way, the two Fc domains of the antibody molecule can pair to form a stable "knob-in-hole" bond. Depending on the expected use, the first and / or second Fc domains of the Format_2 antibody molecule of the present invention may further preferably include mutations that affect antibody effector function, such as LALA mutations, including mutations that reduce ADCC activity.

[0112] In some embodiments, the Format_2 trispecific antibody molecule of the present 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, where, The first heavy chain polypeptide chain, from the N-terminus to the C-terminus, includes the heavy chain variable domain VH, the immunoglobulin CH1 domain, and the Fc domain. The first light chain polypeptide chain includes a light chain variable domain (VL) and an immunoglobulin domain (CL) from the N-terminus to the C-terminus. The second heavy-chain polypeptide chain includes a heavy-chain variable domain VH, an immunoglobulin CH1 domain, and an Fc domain from the N-terminus to the C-terminus. The second light chain polypeptide chain contains a light chain variable domain (VL) and an immunoglobulin domain (CL) from the N-terminus to the C-terminus. Furthermore, the first or second heavy polypeptide chain further comprises an scFv domain compounded directly or preferably via a linked peptide (e.g., (G4S)2 or TS(G4S)2) at the C-terminus of its Fc domain. Here, The VH-CH1 of the first heavy polypeptide chain and the VL-CL of the first light polypeptide chain pair to form the first antibody arm (M1) that binds to the 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 the 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, compounded at the C-terminus of the first or second heavy polypeptide chain, forms a complex component (X2) that binds to the third antigen. Here, the first, second, and third antigens are all different and are independently selected from GPRC5D, BCMA, and CD3.

[0113] In one embodiment, antibody arm M1 is bound to GPCR5D, second antibody arm M2 is bound to CD3, and complex component X2 is bound to BCMA, and in this way, -The first heavy polypeptide chain and the first light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to GPRC5D. -The second heavy polypeptide chain and the second light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to CD3. Here, when complex component X2 is compounded with a first heavy polypeptide chain, the first heavy polypeptide chain further includes an amino acid sequence of scFv that specifically binds to BCMA at its C-terminus, or, preferably, when complex component X2 is compounded with a second heavy polypeptide chain, the second heavy polypeptide chain further includes an amino acid sequence of scFv that specifically binds to BCMA at its C-terminus.

[0114] In another embodiment, antibody arm M1 is bound to BCMA, second antibody arm M2 is bound to CD3, and complex component X2 is bound to GPCR5D, thus, -The first heavy polypeptide chain and the first light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to BCMA. -The second heavy polypeptide chain and the second light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to CD3. Here, when complex component X2 is compounded with a first heavy polypeptide chain, the first heavy polypeptide chain further includes an amino acid sequence of scFv that specifically binds to GPRC5D at its C-terminus, or, preferably, when complex component X2 is compounded with a second heavy polypeptide chain, the second heavy polypeptide chain further includes an amino acid sequence of scFv that specifically binds to GPRC5D at its C-terminus.

[0115] In a further embodiment, antibody arm M1 is bound to GPCR5D, second antibody arm M2 is bound to BCMA, and complex component X2 is bound to CD3, thus, -The first heavy polypeptide chain and the first light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to GPRC5D. -The second heavy-chain polypeptide chain and the second light-chain polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to BCMA. Here, if complex component X2 is compounded with a first heavy polypeptide chain, the first heavy polypeptide chain further includes an amino acid sequence of scFv that specifically binds to CD3 at its C-terminus, or if complex component X2 is compounded with a second heavy polypeptide chain, the second heavy polypeptide chain further includes an amino acid sequence of scFv that specifically binds to CD3 at its C-terminus.

[0116] In one preferred embodiment, the Fab or scFv that specifically binds to GPRC5D belongs to the following group: (a) VH containing the HCDR1-3 sequences of SEQ ID NOs. 1-3 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 4-6 (b) VH containing the HCDR1-3 sequences of SEQ ID NOs. 7-8 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 10-12 (c) VH containing the HCDR1-3 sequences of SEQ ID NOs. 13-15 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 16-18, or (d) VH containing the HCDR1-3 sequences of SEQ ID NOs. 19-21 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 22-24 Includes VH and VL selected from, Preferably, the Fab and the scFv include a VH and VL amino acid sequence pair selected from the following group: SEQ ID NOs: 25 / 26, 27 / 28, 29 / 30, and 31 / 32. Preferably, if scFv is dsscFv, the further includes cysteine ​​substitutions introduced into the VH and VL sequences, for example, a cysteine ​​substitution at position 44 of the VH sequence and position 100 of the VL sequence. In one preferred embodiment, the Fab or scFv that specifically binds to the CD3 is from the following group: (a) VH containing the HCDR1-3 sequences of SEQ ID NOs. 41-43 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46, or (b) VH containing the HCDR1-3 sequences of SEQ ID NOs. 41, 47, and 43, and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46, Includes VH and VL selected from, Preferably, the Fab and scFv comprise a VH and VL amino acid sequence pair selected from the following group: SEQ ID NOs: 48 / 49, SEQ ID NOs: 50 / 49, and more preferably SEQ ID NOs: 48 / 49. Preferably, if scFv is dsscFv, the further includes cysteine ​​substitutions introduced into the VH and VL sequences, for example, a cysteine ​​substitution at position 44 of the VH sequence and position 100 of the VL sequence.

[0117] In a preferred embodiment, the Fab or scFv that specifically binds BCMA is as follows: comprises a VH comprising the HCDR1 to HCDR3 sequences of SEQ ID NOs: 33 to 35 and a VL comprising the LCDR1 to LCDR3 sequences of SEQ ID NOs: 36 to 38, preferably, said Fab and scFv comprise a VH and VL amino acid sequence pair of SEQ ID NO: 39 / 40, and preferably, when the scFv is a dsscFv, it further comprises cysteine substitutions introduced into the VH and VL sequences, for example, cysteine substitutions at position 44 of the VH sequence and position 100 of the VL sequence.

[0118] In some preferred embodiments, the first light chain polypeptide and the second light chain polypeptide each comprise a kappa light chain constant domain CL κ and a lambda light chain constant domain CL λ respectively. 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 the 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 and the second heavy chain polypeptide each comprise a heavy chain constant region CH1 derived from human IgG1, and preferably comprise 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 polypeptide chain and the second heavy polypeptide chain each contain an Fc domain containing a knob or a complementary hole mutation. In one specific embodiment, the Fc domain containing the knob mutation contains T366W, and the Fc domain containing the complementary hole mutation contains T366S, L368A, and Y407V mutations. In one specific embodiment, the Fc domain containing the hole mutation contains 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 containing the complementary knob mutation contains the 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 having a Format_2 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 contains the amino acid sequence of Sequence ID No. 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto.

[0122] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_2 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 contains the amino acid sequence of Sequence ID No. 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto.

[0123] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_2 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 contains the amino acid sequence of Sequence ID No. 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto.

[0124] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_2 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 identical thereto. The second light chain polypeptide chain contains the amino acid sequence of Sequence ID No. 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto.

[0125] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_2 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 contains the amino acid sequence of Sequence ID No. 68, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto.

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

[0127] Therefore, in some preferred embodiments, the present invention provides a triplicate Y-type antibody molecule, the antibody molecule being as follows: (M1:M2-(X1)p)-Fc::Fc, (Format_7) It has the structure, where p=2 in the formula, M1 and M2 represent the first and second antibody arms of the antibody molecule, respectively, and M1 and M2 each contain Fabs that bind to the first and second antigens, respectively. Fc::Fc represents the stem of the antibody molecule, consisting of a paired and dimerized first Fc domain and a second Fc domain, where the first and second antibody arms are ligated directly or via a linking peptide (preferably a hinge region) to the N-terminuses of the first and second Fc domains, respectively. X1 represents a complex component compounded at the C-terminus of the antibody arm, where the complex component includes an scFv that binds to the third antigen, where X1 is compounded at the C-terminus of the Fab light chains of the first and second antibody arms, either directly or via a linked peptide. Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3.

[0128] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_7 structure, where, (a): The antibody arms M1 and M2 of the molecule contain Fab that binds to GPRC5D and Fab that binds to CD3, respectively, and the complex component X1 contains scFv that binds to BCMA, and the molecule has the structure (Fab_GPRC5D:Fab_CD3-(scFv_BCMA)2)-Fc::Fc (for example, the molecule of Examples TS-F7-1,2,5), or (b): The antibody arms M1 and M2 of the molecule each contain Fab that binds to GPRC5D and Fab that binds to BCMA, respectively, and the complex component X1 contains scFv that binds to CD3, and the molecule has the structure (Fab_GPRC5D:Fab_BCMA-(scFv_CD3)2)-Fc::Fc (for example, the molecule of Examples TS-F7-3,4), or (c) The antibody arms M1 and M2 of the molecule each contain Fab that binds to BCMA and Fab that binds to CD3, respectively, and the complex component X1 contains scFv that binds to 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, the GPRC5D antigen-binding site of the present invention having the scFv or Fab structure described above, and in particular, the scFv or Fab having the VH and VL amino acid sequences described above. In any embodiment of the Format_7 antibody molecule of the present invention described above, the Fab or scFv that binds to BCMA may be any suitable Fab or scFv that specifically binds to BCMA, for example, the BCMA antigen-binding site of the present invention having the scFv or Fab structure described above, and in particular, the scFv or Fab having the VH and VL amino acid sequences described above. In any embodiment of the Format_7 antibody molecule of the present invention described above, the antigen-binding site that binds to CD3 may be any suitable scFv or Fab that specifically binds to CD3, for example, the CD3 antigen-binding site of the present invention having the scFv or Fab structure described above, and 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 present invention described above, the antibody molecule may include any antibody stem structure of the present invention applicable to Format_7 as described above. For example, in the Format_7 asymmetric trispecific antibody molecule of the present invention, mutations that promote heterodimerization of the first and second Fc domains, particularly complementary "knob-in-hole" mutations, can be introduced into the first and second Fc domains of the stem, preferably to facilitate the correct pairing of the antibody molecule polypeptide chains. For example, a knob mutation can be introduced into the first Fc domain and a complementary hole mutation into the second Fc domain, or vice versa. In this way, the two Fc domains of the antibody molecule can pair to form a stable "knob-in-hole" bond. Depending on the expected use, the first and / or second Fc domains of the Format_7 antibody molecule of the present invention may further preferably include mutations that affect antibody effector function, such as LALA mutations, including mutations that reduce ADCC activity.

[0131] In some embodiments, the Format_7 trispecific antibody molecule of the present 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, where, The first heavy chain polypeptide chain, from the N-terminus to the C-terminus, includes the heavy chain variable domain VH, the immunoglobulin CH1 domain, and the Fc domain. The first light chain polypeptide chain includes a light chain variable domain (VL) and an immunoglobulin domain (CL) from the N-terminus to the C-terminus. The second heavy-chain polypeptide chain includes a heavy-chain variable domain VH, an immunoglobulin CH1 domain, and an Fc domain from the N-terminus to the C-terminus. The second light chain polypeptide chain contains a light chain variable domain (VL) and an immunoglobulin domain (CL) from the N-terminus to the C-terminus. Furthermore, the first and second light chain polypeptide chains further comprise scFv domains that are compounded directly or preferably via a linked peptide (e.g., (G4S)2 or TS(G4S)2) to the C-terminus of their CL domains. Here, The VH-CH1 of the first heavy polypeptide chain and the VL-CL of the first light polypeptide chain pair to form the first antibody arm (M1) that binds to the 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 the 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 domains compounded at the C-terminuses of the first and second light chain polypeptide chains form a complex component (X1) that binds to the third antigen. Here, the first, second, and third antigens are all different and are independently selected from GPRC5D, BCMA, and CD3.

[0132] In one embodiment, antibody arm M1 is bound to GPCR5D, second antibody arm M2 is bound to CD3, and complex component X1 is bound to BCMA, and in this way, -The first heavy polypeptide chain and the first light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to GPRC5D. -The second heavy polypeptide chain and the second light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to CD3. Furthermore, the first and second light chain polypeptide chains further contain an amino acid sequence of scFv that specifically binds to BCMA at their C-terminus.

[0133] In one embodiment, antibody arm M1 is bound to BCMA, second antibody arm M2 is bound to CD3, and complex component X1 is bound to GPCR5D, and in this way, -The first heavy polypeptide chain and the first light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to BCMA. -The second heavy polypeptide chain and the second light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to CD3. Furthermore, the first and second light chain polypeptide chains further contain an amino acid sequence of scFv at their C-terminus that specifically binds to GPRC5D.

[0134] In one embodiment, antibody arm M1 is bound to GPCR5D, second antibody arm M2 is bound to BCMA, and complex component X1 is bound to CD3, and in this way, -The first heavy polypeptide chain and the first light polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to GPRC5D. -The second heavy-chain polypeptide chain and the second light-chain polypeptide chain each contain the VH and VL amino acid sequences at their N-terminuses, which are Fab antigen-binding sites that specifically bind to BCMA. Furthermore, the first and second light chain polypeptide chains further contain an amino acid sequence of scFv that specifically binds to CD3 at their C-terminus.

[0135] In one preferred embodiment, the Fab or scFv that specifically binds to GPRC5D belongs to the following group: (a) VH containing the HCDR1-3 sequences of SEQ ID NOs. 1-3 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 4-6 (b) VH containing the HCDR1-3 sequences of SEQ ID NOs. 7-8 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 10-12 (c) VH containing the HCDR1-3 sequences of SEQ ID NOs. 13-15 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 16-18, or (d) VH containing the HCDR1-3 sequences of SEQ ID NOs. 19-21 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 22-24 Includes VH and VL selected from, Preferably, the Fab and the scFv include a VH and VL amino acid sequence pair selected from the following group: SEQ ID NOs: 25 / 26, 27 / 28, 29 / 30, and 31 / 32. Preferably, if scFv is dsscFv, the further includes cysteine ​​substitutions introduced into the VH and VL sequences, for example, a cysteine ​​substitution at position 44 of the VH sequence and position 100 of the VL sequence.

[0136] In one preferred embodiment, the Fab or scFv that specifically binds to the CD3 is from the following group: (a) VH containing the HCDR1-3 sequences of SEQ ID NOs. 41-43 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46, or (b) VH containing the HCDR1-3 sequences of SEQ ID NOs. 41, 47, and 43, and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46, Includes VH and VL selected from, Preferably, the Fab and scFv comprise a VH and VL amino acid sequence pair selected from the following group: SEQ ID NOs: 48 / 49, SEQ ID NOs: 50 / 49, and more preferably SEQ ID NOs: 48 / 49. Preferably, if scFv is dsscFv, the further includes cysteine ​​substitutions introduced into the VH and VL sequences, for example, a cysteine ​​substitution at position 44 of the VH sequence and position 100 of the VL sequence.

[0137] In one preferred embodiment, the Fab or scFv that specifically binds to the BCMA is as follows: VH containing HCDR1-3 sequences of sequence numbers 33-35 and VL containing LCDR1-3 sequences of sequence numbers 36-38, Preferably, the Fab and scFv include the VH and VL amino acid sequence pair known as SEQ ID NO: 39 / 40, Preferably, if scFv is dsscFv, the further includes cysteine ​​substitutions introduced into the VH and VL sequences, for example, a cysteine ​​substitution at position 44 of the VH sequence and position 100 of the VL sequence.

[0138] In some embodiments, the first light chain polypeptide chain and the second light chain polypeptide chain are kappa light chain constant domain CL κ Includes. In some embodiments, the first light chain polypeptide chain and the second light chain polypeptide chain have a constant lambda light chain domain CL λ Includes. In one specific embodiment, the light chain constant domain CL κ This includes 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. In one specific embodiment, the light chain constant domain CL λ This includes 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 include a heavy chain constant region CH1 derived from human IgG1, and preferably include 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 polypeptide chain and the second heavy polypeptide chain each contain an Fc domain containing a knob or a complementary hole mutation. In one specific embodiment, the Fc domain containing the knob mutation contains T366W, and the Fc domain containing the complementary hole mutation contains T366S, L368A, and Y407V mutations. In one specific embodiment, the Fc domain containing the knob mutation contains 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 containing the complementary hole mutation contains the 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 having a Format_7 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 identical thereto. The second light chain polypeptide chain contains the amino acid sequence of Sequence 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 having a Format_7 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 contains the amino acid sequence of Sequence 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 having a Format_7 structure comprising or consisting of a first heavy polypeptide chain, a first light polypeptide chain, a second heavy polypeptide chain, and a second light polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 contains the 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 double-chain polypeptide chain contains the 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 contains 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 identical thereto.

[0144] Format_1 In some embodiments, when p=0 and q=1, the antibody molecule of the present invention has the structure (M1:M2)-Fc::Fc-(X2)q, where the antibody arms M1, M2 and complex component X2 each contain 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 different and independently selected from GPRC5D, BCMA, and CD3.

[0145] Therefore, in some preferred embodiments, the present invention provides a triplicate Y-type antibody molecule, the antibody molecule being as follows: (M1:M2)-Fc::Fc-(X2)q, (Format_1) It has the structure, where q=1, M1 and M2 represent the first and second antibody arms of the antibody molecule, respectively, and M1 and M2 each contain scFv that bind to the first and second antigens, respectively. Fc::Fc represents the stem of the antibody molecule, consisting of a paired and dimerized first Fc domain and a second Fc domain, where the first and second antibody arms are ligated directly or via a linking peptide (preferably a hinge region) to the N-terminuses of the first and second Fc domains, respectively. X2 represents a complex component compounded at the C-terminus of the stem, where the complex component includes an scFv that binds to the third antigen, where X2 is compounded at the C-terminus of the first Fc domain directly or via a linked peptide, or at the C-terminus of the second Fc domain. Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3.

[0146] In some embodiments, the present invention provides a triplicate antibody molecule having a Format_1 structure, where, (a): The antibody arms M1 and M2 of the molecule contain scFv that binds to GPRC5D and scFv that binds to CD3, respectively, and the complex component X2 contains scFv that binds to BCMA, and the molecule has the structure (scFv_GPRC5D:scFv_CD3)-Fc::Fc-(scFv_BCMA) (for example, the molecule of Example TS-F1-1), or (b): The antibody arms M1 and M2 of the molecule each contain scFv that binds to BCMA and scFv that binds to CD3, respectively, and the complex component X2 contains scFv that binds to GPRC5D, and the molecule has the structure (scFv_BCMA:scFv_CD3)-Fc::Fc-(scFv_GPRC5D) (for example, the molecule of Example TS-F1-2).

[0147] In some embodiments, the Format_1 trispecific antibody molecule of the present invention comprises or consists of a primary heavy polypeptide chain and a secondary heavy polypeptide chain, where, The first heavy polypeptide chain contains the first scFv and Fc domains from the N-terminus to the C-terminus. The double-chain polypeptide chain contains a second scFv and Fc domain from the N-terminus to the C-terminus. Furthermore, the first or second heavy polypeptide chain further comprises a third scFv domain compounded directly or preferably via a linked peptide (e.g., (G4S)2 or TS(G4S)2) at the C-terminus of its Fc domain. Here, The first scFv of the first heavy polypeptide chain forms the first antibody arm (M1) that binds to the first antigen. The second scFv of the second heavy-chain polypeptide chain forms a second antibody arm (M1) that binds to the 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 compounded at the C-terminus of a first or second heavy polypeptide chain forms a complex component (X2) that binds to the third antigen. Here, the first, second, and third antigens are all different and are independently selected from GPRC5D, BCMA, and CD3.

[0148] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_1 structure comprising or consisting of a first heavy polypeptide chain and a second heavy polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 double-chain polypeptide chain contains 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 identical thereto.

[0149] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_1 structure comprising or consisting of a first heavy polypeptide chain and a second heavy polypeptide chain, where, The first heavy-chain polypeptide chain contains the 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 double-chain polypeptide chain contains 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 identical thereto.

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

[0151] Therefore, in some preferred embodiments, the present invention provides a triplicate symmetric Y-type antibody molecule, the antibody molecule being as follows: (M1:M2-(X2)p)-Fc::Fc-(X2)q, (Format_6) It has the structure, where p=2 and q=2, M1 and M2 represent the first and second antibody arms of the antibody molecule, respectively, and both M1 and M2 contain Fab that binds to the first antigen. Fc::Fc represents the stem of the antibody molecule, consisting of a paired and dimerized first Fc domain and a second Fc domain, where the first and second antibody arms are ligated directly or via a linking peptide (preferably a hinge region) to the N-terminuses of the first and second Fc domains, respectively. X1 and X2 represent complex components compounded to the C-terminus of the antibody arm Fab light chain or the C-terminus of the stem Fc domain, respectively, where complex component X1 contains an scFv that binds to a second antigen, and complex component X2 contains an scFv that binds to a third antigen, where X1 and X2 are compounded to the arm or stem directly or via linked peptides. Here, the first, second, and third antigens are distinct from each other and are independently selected from GPRC5D, BCMA, and CD3.

[0152] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_6 structure, where, (a) The antibody arms M1 and M2 of the molecule contain Fab that binds to GPRC5D, complex component X1 contains scFv of CD3, and complex component X2 contains scFv that binds to BCMA, and the molecule has the structure (Fab_GPRC5D:Fab_GPRC5D-(scFv_CD3)2)-Fc::Fc-(scFv_BCMA)2 (for example, the molecule of Examples TS-F6-1,2), or (b): The antibody arms M1 and M2 of the molecule contain Fab that binds to BCMA, complex component X1 contains scFv of CD3, and complex component X2 contains scFv that binds to GPRC5D, and the molecule has the structure (Fab_BCMA:Fab_BCMA-(scFv_CD3)2)-Fc::Fc-(scFv_GPRC5D)2 (for example, the molecule of Examples TS-F6-3,4).

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

[0154] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_6 structure comprising or consisting of two identical heavy polypeptide chains and two identical light polypeptide chains, where, The heavy-chain polypeptide chain comprises the 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 therewith. The light chain polypeptide chain includes the 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 identical thereto.

[0155] In some embodiments, the present invention provides a trispecific antibody molecule having a Format_6 structure comprising or consisting of two identical heavy polypeptide chains and two identical light polypeptide chains, where, The heavy-chain polypeptide chain comprises the 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 chain polypeptide chain includes the 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 identical thereto.

[0156] III. GPRC5D antibody molecule of the present invention In one embodiment, 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 of the present invention is (i) The sequences of HCDR1, 2, and 3 of the heavy chain variable domain shown in SEQ ID NO: 25, and the sequences of LCDR1, 2, and 3 of the light chain variable domain shown in SEQ ID NO: 26, or (ii) The sequences of HCDR1, 2, and 3 of the heavy chain variable domain shown in SEQ ID NO: 27, and the sequences of LCDR1, 2, and 3 of the light chain variable domain shown in SEQ ID NO: 28, or (iii) Sequences of HCDR1, 2, and 3 of the heavy chain variable domain shown in SEQ ID NO: 29, and sequences of LCDR1, 2, and 3 of the light chain variable domain shown in SEQ ID NO: 30, or (iv) Sequences of HCDR1, 2, and 3 of the heavy chain variable domain shown in SEQ ID NO: 31, and sequences of LCDR1, 2, and 3 of the light chain variable domain shown in SEQ ID NO: 32, or (v) Sequences of HCDR1, 2, and 3 of the heavy chain variable domain shown in SEQ ID NO: 98, and sequences of LCDR1, 2, and 3 of the light chain variable domain shown in SEQ ID NO: 99, or (vi) comprising the sequences of HCDR1, 2, and 3 of the heavy chain variable domain shown in SEQ ID NO: 100, and the sequences of LCDR1, 2, and 3 of the light chain variable domain shown in SEQ ID NO: 101.

[0157] In some other embodiments, the GPRC5D antibody or its antigen-binding fragment of the present invention is (i) Sequences of heavy chain variable domains HCDR1, 2, and 3 shown in Sequence IDs 1 to 6, and sequences of light chain variable domains LCDR1, 2, and 3, or (ii) Sequences of heavy chain variable domains HCDR1, 2, and 3 shown in Sequence IDs 7-12, and sequences of light chain variable domains LCDR1, 2, and 3, respectively, or (iii) the sequences of heavy chain variable domain HCDR1, HCDR2 and HCDR3 and the sequences of light chain variable domain LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 13 to 18, respectively, or (iv) the sequences of heavy chain variable domain HCDR1, HCDR2 and HCDR3 and the sequences of light chain variable domain LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 19 to 24, respectively, or (v) the sequences of heavy chain variable domain HCDR1, HCDR2 and HCDR3 and the sequences of light chain variable domain LCDR1, LCDR2 and LCDR3 set forth in SEQ ID NOs: 13, 110 and 18, respectively; and comprises a combination of CDR sequences selected from the foregoing.

[0158] In some further embodiments, the GPRC5D antibody molecule or antigen-binding fragment thereof of the present invention is:[["END]] (a) 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 (b) 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 (c) a heavy chain variable domain comprising the 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 the 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 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 therewith, 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 therewith, (e) a heavy chain variable domain comprising the 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 therewith, and a light chain variable domain comprising the 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 therewith, (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 therewith, 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 therewith, comprises a combination of VH and VL amino acid sequences selected from the group consisting of:

[0159] Preferably, the GPRC5D antibody molecule or antigen-binding fragment thereof of the present invention is: (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 containing the amino acid sequence shown in SEQ ID NO: 98, and a light chain variable domain containing the amino acid sequence shown in SEQ ID NO: 99, (vi) A combination of VH and VL amino acid sequences selected from a heavy chain variable domain containing the amino acid sequence shown in SEQ ID NO: 100 and a light chain variable domain containing the amino acid sequence shown 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 triplicate antibody. In some preferred embodiments, the GPRC5D antibody of the present invention is a bispecific antibody further comprising an antigen-binding site that binds to CD3, for example, the CD3 antigen-binding site of the present invention described above. In some other preferred embodiments, the GPRC5D antibody of the present invention is a triplicate antibody further comprising an antigen-binding site that binds to BCMA (for example, the BCMA antigen-binding site of the present invention described above) and an antigen-binding site that binds to CD3 (for example, the CD3 antigen-binding site of the present invention described above).

[0161] In some embodiments, the GPRC5D antibody of the present invention is a bispecific antibody that binds to GPRC5D and CD3. Preferably, the antibody here is one of the following groups that bind to CD3: (a) VH containing the HCDR1-3 sequences of SEQ ID NOs. 41-43 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46, or (b) VH containing the HCDR1-3 sequences of SEQ ID NOs. 47, 42, and 43, and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46, It includes VH and VL amino acid sequence pairs selected from, Preferably, the sequence comprises a VH and VL amino acid sequence pair selected from the following group: SEQ ID NOs: 48 / 49 and SEQ ID NOs: 50 / 49. In some cases, the VH and VL sequences include introduced cysteine ​​substitutions, for example, cysteine ​​substitutions at position 44 of the VH sequence and position 100 of the VL sequence, thus forming a disulfide bond between VH and VL.

[0162] Therefore, 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 present invention further provides variants of the bispecific antibody, wherein the variants include amino acid mutations such as amino acid substitutions, deletions, and / or insertions in the VH and VL amino acid sequence pairs that bind to GPRC5D and / or the VH and VL amino acid sequence pairs that bind 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 include combinations 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 VH-VL amino acid sequence pair bound to GPRC5D and / or a VH-VL amino acid sequence pair bound to CD3, for example, a cysteine ​​substitution at position 44 of the VH sequence and position 100 of the VL sequence, thus forming a disulfide bond between VH and VL.

[0165] The present invention further provides fusion proteins and immune complexes (e.g., complexes with toxins or small chemical molecules) containing the GPRC5D antibody of the present invention, as well as pharmaceutical compositions and drug combinations. In a pharmaceutical composition or drug combination, the antibody of the present invention may also contain other therapeutic agents, such as other therapeutic agents applicable to the expected use of the antibody of the present invention, e.g., chemotherapeutic agents, radiotherapeutic agents, tumor suppressor molecules.

[0166] IV. Production and Purification of the Antibody Molecules of the Present Invention In a further embodiment, the present invention provides a method for producing an antibody molecule of the present invention, the method comprising culturing a host cell containing a polynucleotide encoding a polypeptide chain under conditions suitable for the expression of the antibody, and assembling the polypeptide chain under conditions suitable for the assembly of the polypeptide chain into an antibody molecule to produce the antibody.

[0167] When the antibody molecule of the present invention is an asymmetric antibody molecule such as a tripspecific 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, for example, antibody molecules of Format_2 and Format_7, preferably the method for producing the antibody molecule of the present invention comprises the steps of: (i) culturing host cells containing the encoding of the first heavy chain and the first light chain under conditions suitable for expressing the antibody molecule, thereby producing a first parent protein; (ii) culturing host cells containing the encoding of the second heavy chain and the second light chain under conditions suitable for expressing the antibody molecule, thereby producing a second parent protein; and (iii) mixing the first parent protein and the second parent protein in equimolar ratios and assembling them in vitro under appropriate redox conditions to form the antibody. In one specific embodiment, step (iii) includes adding glutathione (GSH) to a mixed solution of purified first and second parental proteins, where preferably the GSH / protein molar ratio is controlled to 500 to 700 times. In one specific embodiment, step (iii) further includes removing the GSH and then performing spontaneous oxidation for a certain period of time (e.g., 2 to 3 hours).

[0168] The polypeptide chains of the antibody molecules of the present invention can be produced, for example, by solid-phase peptide synthesis (e.g., Merrifield solid-phase synthesis) or by recombinant synthesis. In recombinant synthesis, 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 host cells. The polynucleotides can be easily isolated and sequenced using conventional methods. In one embodiment, a vector containing one or more polynucleotides of the present invention, preferably an expression vector, is provided.

[0169] An expression vector can be constructed using methods known to those skilled in the art. The expression vector includes, but is not limited to, viruses, plasmids, cosmids, λ phage or yeast artificial chromosomes (YAC). After an expression vector for expression comprising one or more polynucleotides of the present invention is prepared, the expression vector can be transfected or introduced into an appropriate host cell. To achieve this object, various techniques can be used, for example, protoplast fusion, calcium phosphate coprecipitation, electroporation, retroviral transduction, viral transfection, particle gun, liposome-based transfection or other common techniques.

[0170] In one embodiment, one or more host cells containing the polynucleotide of the present invention are provided. In several embodiments, host cells containing the expression vector of the present invention are provided. As used herein, the term “host cell” refers to any type of cell line capable of producing the antibody molecule of the present invention by engineering methods. Host cells suitable for supporting replication and expression of the antibody molecule of the present invention are known in the art. If necessary, such cells can be transfected or transduced using a specific expression vector, and many cells containing the vector can be cultured and inoculated into a large fermenter to obtain a clinically sufficient amount of the antibody molecule of the present invention. Suitable host cells include prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO) or insect cells. Mammalian cell lines suitable for suspension culture can be used. Examples of usable mammalian host cell lines include SV40-transformed monkey kidney CV1 cell line (COS-7), human fetal kidney cell line (HEK 293 or 293F cells), baby hamster kidney cell line (BHK), monkey kidney cell line (CV1), African green monkey kidney cell line (VERO-76), human cervical cancer cell line (HELA), canine kidney cell line (MDCK), buffalo rat hepatocyte cell line (BRL 3A), human lung cell line (W138), human liver cell line (Hep G2), CHO cell line, NSO cell line, and myeloma cell line lines such as YO, NS0, P3X63, and Sp2 / 0. For an overview of mammalian host cell lines suitable for protein production, see, for example, Yazaki & Wu, Methods in Molecular Biology, Vol. 248 (BKCLo ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In one preferred embodiment, the host cells are CHO, HEK293, or NSO cells. 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, and size exclusion chromatography. The actual conditions for purifying specific proteins also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are known to those skilled in the art. The purity of the antibody molecule of the present invention can be determined by any of the various known analytical methods, which include size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography. The physical / chemical properties and / or biological activity of the antibody molecule relating to this specification can be identified, sorted, or characterized by various known assay methods in the art.

[0171] V. Pharmaceutical compositions, drug combinations, and reagent kits In one embodiment, the present invention provides a composition, for example, a pharmaceutical composition, which comprises an antibody molecule described herein, compounded with a pharmaceutically acceptable vector. As used herein, “pharmaceutically acceptable vector” includes any or all of the following: a physiologically compatible solvent, dispersion medium, isotonic agent, absorption retarder, etc. 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 sole active ingredient in the pharmaceutical composition. In some other embodiments, the pharmaceutical composition may comprise the antibody molecule described herein and one or more therapeutic agents.

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

[0173] The therapeutic agent applied to the pharmaceutical composition and drug combination of the present invention may be any one of the following categories (i) to (iii): (i) a drug that enhances antigen presentation (e.g., tumor antigen presentation), (ii) a drug that enhances effector cell response (e.g., B cell and / or T cell activation and / or recruitment), (iii) a drug that reduces immunosuppression, or (iv) a drug that has a tumor suppressor effect.

[0174] The compositions of the present invention can exist in a variety of forms. These forms include liquid, semi-solid, and solid dosage forms, e.g., liquid solutions (e.g., injectable solutions, infusion solutions), dispersions or suspensions, liposomal preparations, and suppositories. The preferred form is determined by the desired mode of administration and therapeutic use. Generally, the composition is preferred in the form of an injectable solution or an infusion solution. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal (IP), intramuscular) injection. In one 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 administration” refer to modes of administration other than enteral and local administration, and generally involve injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intradermal, intraperitoneal, transtracheal, subcutaneous, and infusion.

[0176] Therapeutic compositions are generally sterile and stable under their manufacturing and storage conditions. Compositions can be prepared in the form of solutions, microemulsions, dispersions, liposomes, or lyophilized products. A sterile injectable solution can be prepared by adding a predetermined amount of the active compound (i.e., antibody molecules) to a suitable solvent, followed by filtration and disinfection. Generally, dispersions are prepared by mixing the active compound with a sterile solvent, which contains the underlying dispersion medium and other components. Coating agents such as lecithin can be used. In the case of dispersions, surfactants can be used to maintain appropriate fluidity in the solution. The absorption of the injectable composition can be extended by including substances that delay absorption, such as monostearates or gelatin, in the composition.

[0177] In some embodiments, the antibody molecules of the present invention are administered orally, for example, with an inert diluent or an edible vector. The antibody molecules of the present invention may be encapsulated in hard or soft gelatin capsules, compressed into tablets, or mixed directly into the target meal. When used for therapeutic purposes by oral administration, the compound may be used in the form of tablets, suppositories, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., mixed with excipients and ingested. For parenteral administration of the antibody molecules of the present invention, the antibody molecules may be coated with a material that prevents their inactivation, or administered together with such material. Furthermore, the therapeutic composition can be administered using medical devices known in the art.

[0178] The pharmaceutical composition of the present invention may contain an antibody molecule described in the present invention in a "therapeutic dose" or a "preventive dose." The "therapeutic dose" refers to the amount necessary to effectively achieve the desired therapeutic outcome at the required dose over the required period. The therapeutic dose varies depending on many factors, such as the disease state, the individual's age, sex, and weight. The therapeutic dose is any amount such that toxicity or harm does not outweigh the beneficial effects of the treatment. Compared to an untreated subject, the "therapeutic dose" preferably suppresses 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%. The inhibitory ability of the antibody molecule of the present invention on the aforementioned measurable parameter (e.g., tumor volume) can be evaluated in an animal model system to demonstrate its effects on human tumors.

[0179] The "prophylactic effective dose" refers to the amount of medication needed to effectively achieve the desired preventive outcome over the required period. Generally, since prophylactic doses are used before the early stages of the disease or at a relatively early stage, the prophylactic effective dose is less than the therapeutic effective dose.

[0180] Reagent kits containing antibody molecules as described herein are also within the scope of the present invention. The kit may include one or more other elements, such as instructions for use, other reagents such as markers or coupling reagents, a pharmaceutically acceptable vector, an apparatus for administration to a subject, or other materials.

[0181] V. Use and Method of the Molecule of the Invention In one embodiment, the present invention provides methods for the in vivo and in vitro use and application of the antibody molecule of the present invention.

[0182] In some embodiments, the use and methods of the present invention relate to the following in vivo and / or in vitro applications of the antibody molecule of the present invention. - The cell surface expresses the GPRC5D antigen, and the cell binds to the GPRC5D antigen with high affinity, such as a KD of less than 10 nM. -Targeting T cells (e.g., CD4+ and / or CD8+ T cells) that express GPRC5D on their surface, particularly GPRC5D-positive tumor cells, -Targeting T cells (e.g., CD4+ and / or CD8+ T cells) that express BCMA on their surface, particularly BCMA-positive tumor cells, - Activating the CD3 downstream signaling pathway in T cells, - To mediate the killing effect of T cells against GPRC5D-positive tumor cells and / or BCMA-positive tumor cells. - Inducing T cells to release cytokines such as TNF-α, IFN-γ, and IL-2. - Suppressing 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, and GPRC5D-positive patient populations with BCMA loss after binding to anti-BCMA molecules. - To avoid recurrence of BCMA escape-mediated tumors, such as recurrence of multiple myeloma.

[0183] In some embodiments, the antibody molecule of the present invention or a pharmaceutical composition containing the antibody molecule of the present invention is used as a drug to treat and / or prevent a disease in an individual, or as a diagnostic tool for a disease, preferably the individual being a mammal, more preferably a human.

[0184] In some embodiments, the present invention provides methods and uses for treating cancer using the antibody molecules of the present invention, particularly the trispecific antibody molecules, where the cancer may be selected from multiple myeloma, melanoma, and B-cell lymphoma. Preferably, the cancer is multiple myeloma. Due to their antigen-binding specificity to BCMA and GPRC5D, the trispecific antibody molecules of the present invention can have a broader patient population for cancer treatment than bispecific antibodies targeting BCMA and CD3, and bispecific antibodies targeting GPRC5D and CD3. In some embodiments, the present invention provides methods for treating BCMA-negative cancer or BCMA-low-expression cancer using the trispecific antibody molecules of the present invention. In some further embodiments, the present invention provides treatment for GPRC5D and CD3 bipositive cancer using the trispecific antibody molecules of the present invention.

[0185] In one embodiment, the present invention provides a diagnostic method for detecting the presence of a relevant antigen in a biological sample, such as serum, semen, urine, or tissue biopsy sample (e.g., derived from an overproliferative 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 described herein, or administering the antibody to a subject, under conditions in which an interaction is expected to occur, and (ii) detecting the formation of a complex between the antibody and the sample (and optionally a control sample). If a complex is formed, it indicates the presence of the relevant antigen and also suggests the suitability or necessity of the treatment and / or prophylaxis described herein.

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

[0187] In some embodiments, the level and / or distribution of the relevant antigen is determined in vivo, for example, by detecting the antibody molecule of the present invention, labeled with a detectable substance, in a non-invasive manner (e.g., by appropriate 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 by detecting the antibody molecule of the present invention, labeled in a detectable manner using, for example, a PET reagent (e.g., 18F-fluorodeoxyglucose (FDG)). In one embodiment, the present invention provides a diagnostic kit comprising an antibody molecule described herein and an instruction manual.

[0188] To aid in understanding the present invention, the following embodiments are described. It is not intended, nor should it be, to interpret these embodiments in any way as limiting the scope of the claims of the present invention. [Examples]

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

[0190] immunity The full-length human GPRC5D sequence was constructed in a pcDNA3.1 vector, and this plasmid was used to immunize Balb\c and Harbour mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) by intramuscular injection every two weeks (50 μg of plasmid per mouse), for a total of three immunizations. Subsequently, a GS-CHO cell line overexpressing human GPRC5D was used for booster immunization by intraperitoneal injection every two weeks (1 × 10⁶ cells per mouse). 7 ), he was immunized a total of two times.

[0191] Cell fusion After the serum titer met the requirements, the mouse spleen was removed to prepare a B lymphocyte suspension, which was then mixed with SP2 / 0 myeloma cells (ATCC) in a 1:2 to 1:1 ratio before electrofusion. The fused cells were transferred from the electrode dish to a 50 mL centrifuge tube and incubated in HAT medium for 1 × 10⁶ days. 4 cells / mL ~ 2 × 10 4 Cells were diluted to a concentration of 100 μL / mL, and 100 μL of cell suspension was added to each well of a 96-well plate. The screening medium was changed on day 7 after fusion, and after culturing for day 10 (or longer, depending on the cell proliferation state), flow cytometry (FACS) detection was performed to screen for positive clones.

[0192] High-throughput screening using hybridoma cells Hybridoma cells specifically expressing anti-GPRC5D antibodies were screened by flow cytometry (FACS). Detected cells (293F-huGPRC5D / GS-CHO-huGPRC5D) were counted, and 1 × 10⁻⁶ cells were identified. 6 Dilution was reduced to 100 μL / mL and added to a 96-well U-bottom plate. Centrifuged at 500 g for 5 min and removed the cell medium. The supernatant from the hybridoma culture in the 96-well plate and the positive control antibody were added to the U-bottom plate to resuspend the cells, 100 μL per well, and allowed to stand on ice for 30 min. Centrifuged at 500 g for 5 min, removed the supernatant, and washed the cells once with PBS. Removed the PBS at 500 g for 5 min. 100 μL of FITC-labeled secondary antibody against goat anti-mouse IgG (Jackson Immunoresear, Cat#115-545-006, diluted 1:500 in PBS) was added to each well containing hybridoma supernatant. 100 μL of PE-labeled secondary antibody against human Fc (BioLegend, Cat#409304) was added to the well containing the positive control antibody (Roche-5E11). The cells were incubated on ice for 30 minutes in the dark. The supernatant was removed and the cells were washed once with PBS at 500 g for 5 minutes. The cells were resuspended in 50 μL of 1× PBS and detected on a FACS instrument.

[0193] The screened positive clones were re-screened using the same method as described above to obtain a total of 82 hybridoma cells 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 cell binding experiments, candidate clones were subcloned using conventional methods. Subsequently, they were detected using the high-throughput screening method described above, and the target-positive wells were extracted and the cells were cryopreserved.

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

[0196] Approximately 5 x 10⁶ cells were newly cultured from each cell line. 6 Individual cells 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). Upstream primers were designed using base sequences located in the FR1 region of the 5' terminal, and downstream primers were designed using bases located in the antibody constant region or FR4 region. Variable region gene fragments of the antibody's light and heavy chains were amplified. These were ligated to a T vector (Mighty TA-cloning Kit reagent kit, Takara), monoclonal sequences were selected and sequenced, and the sequencing results were analyzed and aligned using MEGA7 software.

[0197] Through alignment, clones with correctly paired variable region sequences of the antibody's light and heavy chains were selected. These light and heavy chain variable region gene fragments were then ligated into a pcDNA3.1 vector using homologous recombination enzyme (Exnase® II, catalog number: C112-01) from Nanjing Nuoweizan Corporation. The constant region was selected to be the IgG1 subtype, and expression plasmids for the light and heavy chain antibodies were obtained.

[0198] Next, the light chain plasmid and heavy chain plasmid of the same antibody were mixed in a 1:1 molar ratio, and 293F cells were transfected with polyethyleneimine (PEI) (Polysciences, Cat#23966). After culturing for 5 to 7 days, when cell activity fell below 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 the anti-GPRC5D antibody was detected by flow cytometry. Different concentrations of the antibody were co-incubated for 30 minutes with GS-CHO cells overexpressing human GPRC5D (huGPRC5D GS-CHO), multiple myeloma cells MM1.R, H929, and AMO-1, and other cells expressing human GPRC5D. Next, a fluorescently labeled secondary antibody (APC-mouse anti-human IgG Fc antibody, Biolegend, product number: 409306) was added, and cell fluorescence intensity was detected by flow cytometry using a live / dead yellow dead cell stain (Thermo Fisher, product number: L34967). The curve was fitted using GraphPad Prism 8.0, and the EC50 value was calculated to indicate the antibody's affinity for binding to human GPRC5D.

[0200] Of the detected antibodies, HB15H1G1 (sequence number 25 in the heavy chain variable region, sequence number 26 in the light chain variable region) is a fully humanized antibody, while ch5E12C4 (sequence number 98 in the heavy chain variable region, sequence number 99 in the light chain variable region), ch11B7 (sequence number 100 in the heavy chain variable region, sequence number 101 in the light chain variable region), and ch7F5D4 antibodies (sequence number 27 in the heavy chain variable region, sequence number 28 in the light chain variable region) are chimeric antibodies. The results are shown in Table 1 below. [Table 4]

[0201] 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 a triple-specific antibody (TS). Multispecific antibodies targeting T-cell engagers simultaneously can bind to two tumor-associated antigens on the surface of multiple myeloma (MM) cells, GPRC5D and BCMA, as well as the CD3 receptor on the surface of T cells. 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 (a CD3 antibody with relatively low affinity hzsp34.87 and a CD3 antibody with relatively high affinity hzsp34.24), we designed multispecific antibodies that simultaneously target GPRC5D, BCMA, and CD3.

[0203] As shown in Figure 1, we designed four types of multispecific antibody molecules with different formats, 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). We resolved the heavy chain mispairing of the asymmetric IgG-like bispecific antibodies using the Fc "knob-in-hole" technique, where Fc is the constant region of the IgG1 heavy chain, and we designed exemplary antibodies by introducing L234A and L235A (numbered by Kabat's "EU") amino acid mutations that simultaneously weaken the effector function.

[0204] TS-F2 and TS-F7 are composed of an asymmetrical IgG-like tetramer made from four polypeptide chains, while TS-F6 is composed of a symmetrical IgG-like tetramer made from two polypeptide chains.

[0205] TS-F2 consisted 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 linked 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 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 paired to form the second antigen recognition site, and scFv formed the third antigen recognition site.

[0206] TS-F7 is 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 single-chain antibody scFv linked by a light chain variable domain, an immunoglobulin CL domain, and an artificially synthesized linked 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 single-chain antibody scFv linked by a light chain variable domain, an immunoglobulin CL domain, and an artificially synthesized linked peptide. The heavy chain variable domain of peptide chain #1 and the light chain variable domain of peptide chain #2 pair 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 pair to form the second antigen recognition site, and the two scFvs in peptide chains #2 and #4 are two identical third antigen recognition sites.

[0207] In TS-F2 and TS-F7, the Fc domains of peptide chain #1 and peptide chain #3 each contained the corresponding stable binding mutation for the "nob-in-hole".

[0208] TS-F6 consists of two identical peptide chains, #1, each containing a single-chain antibody scFv linked by a heavy chain variable domain, an immunoglobulin CH1 domain, an Fc domain, and an artificially synthesized linked peptide; and two identical peptide chains, #2, each containing a single-chain antibody scFv linked by a light chain variable domain, an immunoglobulin CL domain, and an artificially synthesized linked peptide. The heavy chain variable domain of peptide chain #1 and the light chain variable domain of peptide chain #2 pair to form the 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 consisted of peptide chain #1 containing a first single-chain antibody scFv and Fc domain, and peptide chain #2 containing a second single-chain antibody scFv and 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 antibody 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 region, light chain variable region, and heavy chain variable region 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 region, light chain variable region, and heavy chain variable region of the antibodies are shown in Table 2. [Table 5] [Table 6] [Table 7] [Table 8]

[0211] We simultaneously designed bispecific antibodies for GPRC5DxCD3 and BCMAxCD3. As shown in Figure 2A, in a rational design, both ends of the bispecific antibody were in Fab form, forming a 1+1 format asymmetric Ig-like structure. The Fc "knob-in-hole" technique resolved the heavy chain mispairing of the asymmetric IgG-like bispecific antibody, where Fc is the constant region of the IgG1 heavy chain, and simultaneously introduced L234A and L235A (numbered by Kabat's "EU") amino acid mutations that weaken the effector function.

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

[0213] Simultaneously, 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 consists of sequences 17, 18, 19, and 20 of sequence numbers 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, and for further cloning and / or expression in host cells, Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the nucleotide sequences encoding the heavy and light chains and insert them into the vector pcDNA3.1, respectively.

[0215] Expression and purification in HEK293 cells: Expi293F cells (purchased from Thermo Fisher Scientific) were subcultured in Expi293F cell medium (purchased from Thermo Fisher Scientific). Cell density was detected the day before transfection, and fresh Expi293 cell medium was subcultured in 2 × 10⁶ cells. 6 Continue culturing after adjusting the cell density to 10 cells / mL, and on the day of transfection, increase the cell density to 3 × 10⁶. 6 The concentration was adjusted to individual cells / mL.

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

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

[0218] The parent proteins generated by recombination for the TS antibodies and bispecific antibodies of the different formats mentioned above are shown below. [Table 9]

[0219] In the case of asymmetric IgG-like antibodies, the two purified parent proteins did not need to be exchanged between solutions, and their respective protein concentrations were >1 mg / mL. They were mixed according to a 1:1 molar ratio and a protein concentration of 1 mg / mL to 10 mg / mL in the mixture. Next, the GSH / protein molar ratio was controlled to 500 to 700 times, and an appropriate amount of GSH was added so that the final GSH concentration was >5 mM. Finally, the pH of the mixture was adjusted to 8.0 to 8.5 with 1M Arg at pH 10.0. The final Arg concentration was >= 50 mM. The reaction solution was left overnight at room temperature for no more than 24 hours. The next day, the reaction solution that had reacted overnight was replaced to remove the GSH. The buffer for the solution exchange may be the 0.2M PB solution (Ph 6.0) used for the recombinant reaction, which does not contain GSH. After 2 to 3 hours of spontaneous oxidation, the solution may be purified with monoS (GE Cat. 17516801).

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

[0221] The symmetrical and asymmetrical antibodies produced by the above method were recovered and purified by affinity chromatography and ion exchange chromatography. Size exclusion chromatography (SEC) was used to detect the purity of the samples in each sub-tube collected by chromatography. Based on the SEC results, samples from sub-tubes with a purity greater than 95% were combined. The purified bispecific antibody solution was centrifuged in a 15 mL ultrafiltration centrifuge tube at 4500 rpm for 30 minutes. After diluting the protein with PBS, centrifugation was continued, and the buffer was changed by centrifugation at 4500 rpm for 30 minutes. This procedure was repeated several times. The antibodies with changed buffers were combined, and the antibody concentrations were measured. Furthermore, the components and content of the multispecific antibodies were qualitatively and quantitatively analyzed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0222] Example 6: Measurement of Triple-Specific Antibody Affinity The binding affinity of the antigen-binding sites derived from the humanized antibodies hz5E12.1.P1 and hz11B7.5 to GPRC5D was investigated for exemplary trispecific antibodies. Using the same method as in Example 3, different concentrations of trispecific antibodies were co-incubated with huGPRC5D GS-CHO cells for 30 minutes, then a fluorescently labeled secondary antibody was added. Cell fluorescence intensity was detected by flow cytometry (BD), and the curves were fitted using GraphPad Prism 8.0. The EC50 value was calculated to indicate the affinity of the antibody to bind to human GPRC5D. The results are shown in Table 5 below. [Table 10]

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

[0224] Probe sensors (Fortebio, product number 18-5019) containing conjugated streptavidin protein (SA) or anti-human-Fc (AHC) were pre-moistened 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 and each diluted sample (100 nM) were added to 96-well black plates (Greiner, product number 655209). The probes and samples were placed on Octet plates (Fortebio, Red96e). Data Acquisition 10.0 was launched, "New Kinetic Experiment" was selected, seeding was performed according to the sample location, the sensor locations were selected, and the execution steps and times were set to: Baseline 60s, Loading 250s, Baseline 100s, Association 600s, and Dissociation 600s. The experiment rotation speed was 1000 rpm and the temperature was 30°C.

[0225] The results were analyzed using the "Data Analysis 10.0" software, the buffer reference channel was subtracted, a 1:1 binding was selected, and the data was fitted to calculate the Kon, Koff, and KD values ​​for the exemplary antibody.

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

[0227] In the experiments described in the measurement method above, the affinity of exemplary antibodies was detected using human CD3D&E heterodimer antigen (ACRO Biosystems) and monkey CD3D&E heterodimer antigen (ACRO Biosystems), and the results are shown in Tables 8 and 9. Simultaneously, the binding affinity of GPRC5D×CD3 bispecific antibodies, hz5E12.1-P1 / SP34.24 and hz5E12.1-P1 / SP34.87 to human / monkey CD3D&E was investigated. [Table 13] [Table 14]

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

[0229] In Jurkat NFAT luciferase cells and MM tumor cells, the expression of Jurkat cell luciferase was observed after adding exemplary antibodies at different concentrations. [Table 15]

[0230] In short, first, sample dilution: The antibody was diluted to a concentration of 100 nM using RPMI medium 1640 (5% FBS), and then gradient diluted 5-fold. 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 measured using Jurkat NFAT 2x10⁻¹⁰. 6The cell / mL ratio was adjusted, and 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, followed by 30 μL of gradient-diluted sample, and then 45 μL of Jurkat NFAT cells. The 96-well plate was then placed in a 37°C, 5% CO2 incubator to continue culturing. After a set period of incubation, the culture plate and cell culture plate were removed and left at room temperature for 5 minutes. 80 μL of Bio-Glo (Promega, G7940) was added to each well, and the plates were incubated in the dark for 10 minutes. Fluorescence values ​​were read using SpectraMax i3 (MOLECULAR DEVICES). Curves were fitted using GraphPad Prism 8.0, and EC50 values ​​were calculated to compare the T-cell activating activity of exemplary antibodies. Results are presented as the ratio of multiples of experimental group readings to readings of the control hIgG1 group.

[0231] result: TS-F2-1 and TS-F2-2 Under conditions of co-culturing H929 cells with Jurkat cells (effector cell-to-tumor cell ratio of 5:1) for 16 hours, and under conditions of co-culturing L363 cells with Jurkat cells (effector cell-to-tumor cell ratio of 5:1) for 16 hours, both TS-F2-1 and TS-F2-2 significantly increased Jurkat cell luciferase expression with increasing antibody concentration (Figures 3A and 3B), and their agonist activity was higher than that of TS-F1, the control group GPRC5DxCD3 biantibody, and BCMAxCD3 biantibody. The specific EC50 values ​​of the detected antibodies are shown in Table 10. Here, the 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 conditions of co-culturing H929 cells with Jurkat cells (effector cell-to-tumor cell ratio of 5:1) for 16 hours, L363 cells with Jurkat cells (effector cell-to-tumor cell ratio of 5:1) for 16 hours, and U266 cells with Jurkat cells (effector cell-to-tumor cell ratio of 5:1) for 16 hours, TS-F2-3 significantly increased Jurkat cell luciferase expression with increasing antibody concentration (Figures 4A, 4B, and 4C), and its agonist activity was significantly higher than that of the TS-F6 antibody, and higher than that of the control group GPRC5DxCD3 biantibody (ch7F5D4 / hzsp34.24) and BCMAxCD3 (46758) biantibody. The specific EC50 values ​​of the detected antibodies are shown in Table 11. The 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] GS-CHO (huGPRC5D-GS-CHO) cells overexpressing the constructed huGPRC5D were co-cultured with Jurkat cells for 16 hours, and exemplary antibodies were co-cultured to simulate multiple myeloma cell-mediated T cell activation expressing only GPRC5D. 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 significantly increased the expression of luciferase in Jurkat cells as the antibody concentration increased (Figure 4D), and its agonist activity was higher than that of TS-F6 antibody, the control group GPRC5DxCD3 biantibody, and BCMAxCD3 biantibody (46758). Specific EC50 values ​​are shown in Table 11. [Table 17]

[0234] TS-F2-4 and TS-F2-5 Under conditions of co-culturing H929 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, Under conditions of co-culturing L363 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, Under conditions of co-culturing 8226 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, Under conditions of co-culturing U266 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, Both TS-F2-4 and TS-F2-5 significantly increased Jurkat cell luciferase expression with increasing antibody concentration (Figures 5A, 5B, and 5C), and their agonist activity was higher than that of the control group GPRC5DxCD3 and BCMAxCD3 biantibodies (38456 / hzsp34.24) and 46758 biantibodies. Specific EC50 values ​​are shown in Table 12. Here, the exemplary antibody TS-F2-4 consists of hz5E12.1.P1 (GPRC5D antibody), hzsp34.24 (high-affinity CD3 antibody), and 38456 (BCMA antibody), while the exemplary antibody TS-F2-5 consists of hz5E12.1.P1 (GPRC5D antibody), hzsp34.87 (low-affinity CD3 antibody), and 38456 (BCMA antibody). [Table 18]

[0235] Under conditions of co-culturing MM1.S cells and Jurkat cells (with an effector cell-to-tumor cell ratio of 10:1) for 5 hours, and under conditions of co-culturing ARD cells and Jurkat cells (with an effector cell-to-tumor cell ratio of 10:1) for 5 hours, TS-F2-4 and TS-F2-5 significantly increased Jurkat cell luciferase expression with increasing antibody concentration (Figure 5E), and their agonist activity was higher than that of the control groups GPRC5DxCD3 biantibody and BCMAxCD3 biantibody 46758. Specific EC50 values ​​are shown in Table 13. [Table 19]

[0236] TS-F7-1 and TS-F7-2 Under conditions of co-culturing H929 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, Under conditions of co-culturing L363 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, Under conditions of co-culturing 8226 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, Under conditions of co-culturing U266 cells and Jurkat cells (with an effector cell to tumor cell ratio of 5:1) for 5 hours, TS-F7-1 and TS-F7-2 significantly increased Jurkat cell luciferase expression with increasing antibody concentration (Figures 6A, 6B, 6C, and 6D), and their agonist activity was higher than that of the control group GPRC5DxCD3 biantibody and BCMAxCD3 biantibody 46758. Specific EC50 values ​​are shown in Table 14. Here, the exemplary antibody TS-F7-1 consists of hz5E12.1.P1 (GPRC5D antibody), hzsp34.24 (CD3 antibody), and 38456 (BCMA antibody), while the exemplary antibody TS-F7-2 consists of hz11B7.5 (GPRC5D antibody), hzsp34.87 (CD3 antibody), and 38456 (BCMA antibody). [Table 20]

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

[0238] Example 8: Killing experiment of anti-GPRC5DxBCMAxCD3 antibody against human multiple myeloma cells. The exemplary antibody-mediated T cell killing ability against human multiple myeloma cells was detected under conditions of co-culture of PBMCs and GPRC5D and / or BCMA-positive MM cells using the lactate dehydrogenase (LDH) method. When anti-GPRC5DxBCMAxCD3 multiantibodies bind to GPRC5D and / or BCMA on the surface of MM cells and simultaneously to CD3E on the surface of primary T cells, they can stimulate T cell activation and mediate tumor cell killing by cross-linking with tumor-associated antigen (GPRC5D and / or BCMA)-dependent T cells. 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 lysed in a 37°C water bath, added to 9 mL of serum-free medium, centrifuged at 300 g for 4 mins, discarded the supernatant, and resuspended in 10% FBS phenol red-free 1640 medium, adjusting the cell density to 4 × 10⁶. 6 The cells were adjusted to cell / mL. A fixed volume of log-phase H929 cells was collected, centrifuged, and the tumor cell density was adjusted to 2 × 10⁶ in 10% FBS phenol red-free 1640 medium. 5 The concentration was adjusted to cells / mL. According to the table above, the antibody was diluted to 100 nM in 10% FBS phenol red-free 1640 medium, using a 5-fold gradient dilution from 5 μM. 100 μL of tumor cells, 50 μL of the gradient-diluted sample, and 50 μL of PBMC cells were added to a 96-well cell culture plate (U-shaped). Simultaneously, a control well was prepared and replenished with medium to 200 μL / well. The cells were incubated at 37°C in a CO2 incubator for 16 hours. After 16 hours, the cell culture plate was removed and left at room temperature for 5 minutes. 10% Lysis Solution was added to each TM well, and the plate was fused in the dark for 15 minutes. The cell culture plate was centrifuged at 400 g for 5 minutes, and 50 μL of the supernatant was taken and transferred to a new flat-bottom 96-well plate. 50 μL of LDH chromogenic reagent was added, and the plate was allowed to develop color at room temperature for 30 minutes in the dark. 50 μL of stop solution was then added. Absorbance was read using a plate reader, and cell toxicity was calculated based on the absorbance. For subsequent cytokine detection, an additional 90 μL of supernatant was taken into a new 96-well plate (V-shaped). Each well of the cell culture plate was washed once with 200 μL of FACS buffer, the liquid was removed after centrifugation, 45 μL of staining solution (prepared with FACS buffer) was added to each well, and the cells were incubated at 4°C for 20 minutes in the dark. After washing once with 200 μL of FACS buffer and resuspending with 150 μL of FACS buffer, on-board detection was performed.

[0241] Cytokine detection by CBA: To obtain standard 0 (std 0), the standard was dissolved in 2 mL of assay diluent, then 8 wells std 1-8 were gradient diluted 2-fold, and std 9 was used as an assay diluent blank control. Each bead was vortexed for 5-10 seconds, 322 μL was aspirated from each, then 2254 μL of assay diluent was added, and the mixture was vortexed to ensure homogeneity. A 96-well V-shaped plate was prepared, 50 μL of the homogeneously mixed beads were added to each well, 30 μL of assay diluent was added to each well, and then 30 μL of the sample was added to each well, i.e., all samples were diluted 2-fold. PE detection reagent was used after being diluted 1:1 with assay diluent. 50 μL of std or sample and PE detection reagent were added to each well, the plate was sealed with Parafilm, and covered with aluminum foil. The plate was shaken in a plate shaker at a rotation speed of 1100 rpm at room temperature for 5 minutes. Next, the samples were incubated at room temperature for 3 hours. After incubation, 120 μL of wash buffer was added to each well, and the mixture was centrifuged at 300 g for 5 minutes. The supernatant was aspirated. 120 μL of wash buffer was added to resuspend the samples, and they were detected by flow cytometry.

[0242] Experimental results: When detected by flow cytometry, the exemplary antibody was found to be able to activate primary T cells in a dose-dependent manner (Figures 8A and 8B), and a certain correlation was found between the degree of T cell activation and CD3 affinity: the higher the affinity of the anti-CD3 antibody, the stronger its ability to activate T cells, thereby mediating the killing effect of PBMCs on H929 cells (Figure 9). The release levels of various cytokines associated with the killing process are shown in Figures 11A to 11D.

[0243] Example 9: Antitumor efficacy of exemplary antibodies in an in vivo humanized mouse model of H929 tumor. Female NOG mice (35-41 days old) were purchased from Beijing Weitongda Laboratory Animal Technology Co., Ltd. They were SPF grade. After receiving the mice, they were acclimatized and isolated for 7 days before the start of the study. H929 cells were routinely subcultured, centrifuged to collect cells, and dispersed in PBS for subsequent in vivo experiments. 5 × 10 6 H929 cells, with the hair removed, were inoculated into the right dorsal ventral region of NOG mice at a dose of 200 μL per mouse. On day 5 after H929 cell inoculation, 5 × 10⁶ cells were obtained. 6 PBMC cells were intravenously injected into mice at a dose of 200 μL per mouse.

[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. A total of three doses were administered, once every 7 days. Tumor volume and body weight of the mice were monitored twice a week. Tumor volume measurement: The maximum long axis (L) and maximum short axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L × W 2 The calculation was performed using the formula / 2. Body weight was measured using an electronic balance. Throughout the study period, mice were euthanized when the tumor reached the endpoint or when the mouse's body weight loss was >20%. Tumor size was statistically analyzed.

[0245] Experimental results The tumor growth curve is shown in Figure 12, and the exemplary antibody can significantly suppress the growth of H929 cells. No weight loss was observed in any of the mouse groups administered simultaneously.

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

[0247] 1 x 10 5 Individual MM cells were added to a 96-well plate, with GS-CHO-huGPRC5D and GS-CHO-huBCMA cells serving as positive controls and negative controls for each other. Centrifugation was performed at 400g for 5 minutes, 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 hour. Negative control: IgG1. QIFIKIT / Beads preparation: 100 μL of homogeneously mixed beads (Vial 1 and Vial 2) were added to each. 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 homogeneously mixed, and incubated at 4°C in the dark for 45 minutes. After adding 200 μL of FACS buffer and washing three times, 100 μL of PBS was added, mixed uniformly, and detected by flow cytometry.

[0248] Experimental Results: As shown in Figure 13, the different MM cells tested had different surface GPRC5D and BCMA expression levels. As shown in Figures 5A-5F, 6A-6D, and 7, the evaluated Format 2 and Format 7 tripspecific antibodies induced favorable biological activity in various MM cells (H929, L363, 8226, U266, MM1.S, and ARD) with different GPRC5D and BCMA expression levels, compared to control GPRC5D / CD3 and BCMA / CD3 biantibodies. This suggests that by targeting GPRC5D and BCMA in combination, the tripspecific antibodies of the present invention can cover both GPRC5D-positive and BCMA-positive tumor patients, improving coverage for patients with multiple tumors, while simultaneously avoiding tumor recurrence due to the loss of single antigens such as BCMA, thereby improving therapeutic efficacy.

Claims

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

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

3. The antibody or antigen-binding fragment according to claim 2, wherein the VH and VL sequences include cysteine ​​substitutions 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 according to any one of claims 1 to 3, wherein the antigen-binding fragment is Fab, scFv, or dsscFv.

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

6. The antibody or antigen-binding fragment according to 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 sites that bind to the CD3 include VH and VL selected from the group consisting of the following: (a) VH containing the HCDR1-3 sequences of SEQ ID NOs. 41-43 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46 (b) VH containing the HCDR1-3 sequences of SEQ ID NOs. 47, 42, and 43, and VL containing the LCDR1-3 sequences of SEQ ID NOs. 44-46, (c) VH containing the amino acid sequence shown in SEQ ID NO: 48 and VL containing the amino acid sequence shown in SEQ ID NO: 49 (d) VH containing the amino acid sequence shown in SEQ ID NO: 50 and VL containing the amino acid sequence shown in SEQ ID NO: 49, (e) any of (a) to (d) above, wherein the antigen-binding site that binds to the CD3 is dsscFv, the VH contains a cysteine ​​substitution at position 44, and the VL contains a cysteine ​​substitution at position 100. The antibody or antigen-binding fragment according to claim 6.

8. The antibody or antigen-binding fragment according to claim 6, comprising the following antigen-binding site that binds to the BCMA: (a) VH containing the HCDR1-3 sequences of SEQ ID NOs. 33-35 and VL containing the LCDR1-3 sequences of SEQ ID NOs. 36-38 (b) VH containing the amino acid sequence shown in SEQ ID NO: 39 and VL containing the amino acid sequence shown in SEQ ID NO: 40, or (c) The embodiment of (a) or (b) above, wherein the antigen-binding site that binds to the BCMA is dsscFv, the VH contains a cysteine ​​substitution at position 44, and the VL contains a cysteine ​​substitution at position 100.

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, where, The first heavy chain polypeptide chain comprises a heavy chain variable domain VH, an immunoglobulin CH1 domain, and an Fc domain from the N-terminus to the C-terminus. The first light chain polypeptide chain includes a light chain variable domain (VL) and an immunoglobulin (CL) domain from the N-terminus to the C-terminus. The second heavy chain polypeptide chain comprises a heavy chain variable domain VH, an immunoglobulin CH1 domain, and an Fc domain from the N-terminus to the C-terminus, and The second light chain polypeptide chain includes a light chain variable domain (VL) and an immunoglobulin (CL) domain from the N-terminus to the C-terminus. Here, the first heavy-chain polypeptide chain or the second heavy-chain polypeptide chain further comprises an scFv domain compounded to the C-terminus of its Fc domain either directly or via a linked peptide. Here, The VH-CH1 of the first heavy polypeptide chain pair and the VL-CL of the first light polypeptide chain pair to form the first antibody arm (M1) that binds to the 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 the 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 compounded at the C-terminus of the first or second heavy polypeptide chain forms a complex component (X2) that binds to the third antigen. Here, the first, second, and third antigens are all different and are independently selected from GPRC5D, BCMA, and CD3. The antibody or antigen-binding fragment 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, where, (a) The first heavy polypeptide chain comprises the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the first light polypeptide chain comprises the amino acid sequence of SEQ ID NO: 76, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the second heavy polypeptide chain comprises the 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 identical thereto; the second light 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 identical thereto; (b) The first heavy polypeptide chain comprises the 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 polypeptide chain comprises the 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 polypeptide chain comprises the 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 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; (c) The first heavy polypeptide chain includes the 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 polypeptide chain includes the 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 polypeptide chain includes the 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; the second light polypeptide chain includes 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; or (d) The first heavy polypeptide chain includes the amino acid sequence of SEQ ID NO: 79, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the first light polypeptide chain includes the amino acid sequence of SEQ ID NO: 80, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the second heavy polypeptide chain includes the amino acid sequence of SEQ ID NO: 77, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the second light polypeptide chain includes 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 identical thereto; The antibody or antigen-binding fragment according to 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, where, The first heavy chain polypeptide chain comprises a heavy chain variable domain VH, an immunoglobulin CH1 domain, and an Fc domain from the N-terminus to the C-terminus. The first light chain polypeptide chain includes a light chain variable domain (VL) and an immunoglobulin (CL) domain from the N-terminus to the C-terminus. The second heavy chain polypeptide chain comprises a heavy chain variable domain VH, an immunoglobulin CH1 domain, and an Fc domain from the N-terminus to the C-terminus, and The second light chain polypeptide chain includes a light chain variable domain (VL) and an immunoglobulin (CL) domain from the N-terminus to the C-terminus. Here, the first light chain polypeptide chain and the second light chain polypeptide chain further comprise an scFv domain compounded to the C-terminus of their CL domain either directly or via a linked peptide. Here, The VH-CH1 of the first heavy polypeptide chain and the VL-CL of the first light polypeptide chain pair to form the first antibody arm (M1) that binds to the 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 the 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 domains compounded at the C-terminuses of the first and second light chain polypeptides form a complex component (X1) that binds to the third antigen. Here, the first, second, and third antigens are all different and are independently selected from GPRC5D, BCMA, and CD3. The antibody or antigen-binding fragment 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, where, (a) The first heavy polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the first light polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the second heavy polypeptide chain comprises the 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 identical thereto; the second light polypeptide chain comprises the amino acid sequence of SEQ ID NO: 89, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; (b) The first heavy polypeptide chain comprises the 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 polypeptide chain comprises the 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 polypeptide chain comprises the 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 polypeptide chain comprises the 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 polypeptide chain includes the amino acid sequence of SEQ ID NO: 92, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the first light polypeptide chain includes the 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 identical thereto; the second heavy polypeptide chain includes the amino acid sequence of SEQ ID NO: 94, or an amino acid sequence having at least 90%, 92%, 95%, 97%, 98%, 99%, or more identical thereto; the second light polypeptide chain includes 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 identical thereto; The antibody or antigen-binding fragment according to claim 12.

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

15. A vector comprising the polynucleotide described in claim 14.

16. A host cell comprising the polynucleotide described in claim 14 or the vector described in claim 15.

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

18. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition according to claim 18, for use as an agent for treating and / or preventing cancer in an individual, or as a diagnostic tool for diagnosing cancer.

20. The pharmaceutical composition according to claim 19, wherein the cancer is multiple myeloma (MM).

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