Manufacturing and applications of anti-GPRC5D / BCMA / CD3 trispecific antibodies

Abstract

The present invention relates to a triplicate antigen-binding protein, particularly a triplicate antibody that specifically binds to two tumor antigens, GPRC5D and BCMA, and the T cell surface antigen CD3, a pharmaceutical composition containing the same, a method for preparing the same, and the use of the same.

Description

[Technical Field]

[0001] [Cross-reference of related applications] This application is based on and claims priority to China Patent Application No. 202310664900.5 filed on June 6, 2023, and China Patent Application No. 202410713270.0 filed on June 4, 2024, the entire contents of these applications are incorporated herein by reference.

[0002] The present invention relates to a triplicate antigen-binding protein, particularly a triplicate antibody that specifically binds to two tumor antigens, GPRC5D and BCMA, and the T cell surface antigen CD3, a pharmaceutical composition containing the same, a method for preparing the same, and the use of the same. [Background technology]

[0003] Bispecific antibodies (BsAbs) have been used in tumor therapy. These antibodies form synapses between cytotoxic T lymphocytes and tumor cells, inducing the destruction of tumor cells. BsAbs generally involve dual targeting of tumor-associated antigens (TAAs) and T-cell surface antigens (also known as T-cell engaging antigens (TEAs)). However, BsAb-based therapeutic strategies usually depend on the distribution of tumor-associated antigens in the target tumor cells. As a result, therapeutic selectivity arises for the patient population, limiting the applicability of treatment. Furthermore, therapeutic approaches targeting a single TAA site have been shown to cause recurrence due to tumor escape mechanisms, thereby limiting therapeutic efficacy.

[0004] The development of triplicate and / or quadruplicate antibodies has been proposed to effectively recruit immune cells to tumor sites and improve therapeutic efficacy. For this reason, various polyvalent and multispecific antibody formats have been described in recent years. However, due to the diversity of requirements regarding therapeutic function and behavior, it has become clear that there is no single "optimal form" applicable to most desirable molecular combinations. Therefore, achieving multispecificity requires considering various factors, such as the spatial distribution or size of each target antigen, and the expression density of tumor-associated antigens on the tumor cell surface. In many cases, an antibody format effective for a particular target antigen combination should be identified by creating various formats and comparing their functionality.

[0005] The B-cell maturation antigen (BCMA, i.e., CD269 or TNFRSF17) is a member of the tumor necrosis factor receptor superfamily (TNFRSF). BCMA is a type III transmembrane protein with a cysteine-rich domain (CRD) that forms a ligand-binding motif in its extracellular domain (ECD), and is unique to the TNFR superfamily. BCMA ligands include B-cell activator (BAFF) and proliferation-inducing ligand (APRIL), with APRIL binding to BCMA with high affinity and promoting tumor cell proliferation.

[0006] BCMA has been found to be highly expressed in multiple myeloma (MM) cells in preclinical models and human tumors, upregulating normal and irregular NF-κB signaling, promoting MM cell proliferation, survival, and adhesion, and further inducing osteoclast activation, angiogenesis, metastasis, and immunosuppression. BCMA expression is an important marker for diagnosing MM. Furthermore, elevated soluble BCMA (sBCMA) concentrations in the serum of MM patients are directly proportional to the number of MM cells in the bone marrow, and changes in these concentrations are closely correlated with the prognosis and treatment response of MM. Multiple myeloma (also known as plasmacytoma or Karel's disease) is a refractory B-cell malignancy characterized by abnormal proliferation of plasma cells. Given that BCMA is expressed only in plasma cells and not in naive or memory B cells, BCMA is an important target in the treatment of B-cell malignancies, particularly multiple myeloma. The development of antibodies targeting BCMA, particularly multispecific antibodies, is in demand in this industry.

[0007] Prior art (e.g., WO2022174813A1) discloses a tripspecific antibody that specifically binds to CD3, BCMA, and GPRC5D. In this molecular structure, the BCMA antigen-binding site is in the form of scFv and is located at the C-terminus of Fc. However, this antibody has several drawbacks: low purification yield and high aggregation; insufficient antigen affinity and spatial arrangement of the BCMA antigen-binding site scFv, resulting in extremely weak BCMA-mediated tumor cell killing; and the BCMA antigen-binding site contains some hotspot areas that are problematic from a pharmaceutical suitability standpoint.

[0008] Therefore, there is a need for a novel anti-GPRC5D / BCMA / CD3 trispecific antibody that overcomes the above disadvantages, has superior tumor-killing efficacy, is easy to purify, and has excellent pharmacovigilance. [Overview of the project]

[0009] The present invention provides a triplicate antibody comprising a first antigen-binding site that specifically binds to BCMA, and second and third antigen-binding sites that specifically bind to other antigens.

[0010] In some embodiments, the second antigen-binding site specifically binds to CD3, and the third antigen-binding site specifically binds to GPRC5D.

[0011] In some embodiments, the triplicate antibodies of the present invention prevent light chain mispairing by applying charge mutations and disulfide bond remodeling mutations. In some embodiments, the first antigen-binding site that specifically binds to BCMA in the triplicate antibodies of the present invention is Fab.

[0012] In some embodiments, the second antigen-binding site that specifically binds to CD3 is Fab.

[0013] In some embodiments, antigen-binding sites such as Fab that specifically bind to BCMA are located on the N-terminal side of antigen-binding sites such as Fab that specifically bind to CD3. In some embodiments, when the triplicate antibody forms an immune synapse for T cell killing, the triplicate antibody of the present invention allows T cells to get closer to tumor cells than the triplicate antibody known in the prior art (for example, the triplicate antibody disclosed in WO2022174813A1, particularly in which scFv that specifically binds to BCMA is located at the C-terminus of Fc), resulting in more potent T cell killing.

[0014] In some embodiments, the triplicate antibody of the present invention has a structure in which the first antigen-binding site that specifically binds to BCMA can block the function of CD3, thereby avoiding systemic nonspecific T cell activation and subsequent toxicity in the absence of tumor antigens.

[0015] In some embodiments, the triplicate antibody of the present invention has improved pharmacovigilance and / or reduced immunogenicity by having a specific antigen-binding site that specifically binds to BCMA.

[0016] Therefore, the trispecific antibody of the present invention has one or more, or all, of the following advantages compared to the trispecific antibody disclosed in WO2022174813A1.

[0017] i) Improved affinity for BCMA.

[0018] ii) Improved BCMA-mediated tumor killing.

[0019] iii) Reduction of the tendency of antibody molecules to aggregate and / or improvement of the purification yield.

[0020] iv) Enhancement of T cell killing ability.

[0021] v) Reduction of non-specific cytotoxicity (e.g., avoidance of activation of systemic non-specific T cells and subsequent toxicity in situations where tumor antigens are absent).

[0022] vi) Reduction of the immunogenicity of the antibody.

[0023] vii) Improvement of pharmaceutical suitability.

Brief Description of the Drawings

[0024] The preferred embodiments of the present invention described in detail below are better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, currently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangements and means of the embodiments shown in the drawings. [Figure 1] FIG. 1 shows an exemplary structure of a trispecific antibody, FIG. 1A corresponds to the B3 and B3b formats, FIG. 1B corresponds to the B5 structure, and FIG. 1C corresponds to the F2 format. [Figure 2A] FIG. 2A shows the cell-based affinity-1 of an exemplary antibody for human BCMA (GS-CHO-hBCMA). [Figure 2B] FIG. 2B shows the cell-based affinity-2 of an exemplary antibody for human BCMA (GS-CHO-hBCMA). [Figure 2C]Figure 2C shows the cell-based affinity-1 of exemplary antibodies against cynomolgus monkey BCMA (GS-CHO-cynoBCMA). [Figure 2D] Figure 2D shows the cell-based affinity-1 of exemplary antibodies against human GPRC5D (GS-CHO-hGPRC5D). [Figure 2E] Figure 2E shows the cell-based affinity-2 of exemplary antibodies against human GPRC5D (GS-CHO-hGPRC5D). [Figure 2F] Figure 2F shows the cell-based affinity-1 of exemplary antibodies against cynomolgus monkey GPRC5D (GS-CHO-cynoGPRC5D). [Figure 2G] Figure 2G shows the cell-based affinity -1 of exemplary antibodies against human CD3 (Jurkat). [Figure 2H] Figure 2H shows the cell-based affinities of exemplary antibodies against human CD3 (CD8+ T cells and CD4+ T cells). [Figure 2I] Figure 2I shows the cell-based affinity of exemplary antibodies against 293T cells expressing the mutant BCMA antigen. [Figure 2J] Figure 2J shows the cell-based affinity of exemplary antibodies against GS CHO cells expressing the mutant BCMA antigen. [Figure 3A] Figure 3A shows the toxic effect of PBMCs on H929 (GPRC5D knockout) cells mediated by the exemplary antibody-1. [Figure 3B] Figure 3B shows the toxic effect of PBMCs on H929 (GPRC5D knockout) cells mediated by the exemplary antibody-2. [Figure 3C] Figure 3C shows the toxic effect of exemplary antibodies on PBMCs against H929 (GPRC5D knockout) cells -3. [Figure 3D] Figure 3D shows the toxic effect of PBMCs on H929 (BCMA knockout) cells mediated by the exemplary antibody-1. [Figure 3E] Figure 3E shows the toxic effect of PBMCs on H929 (BCMA knockout) cells mediated by the exemplary antibody-2. [Figure 3F] Figure 3F shows the toxic effect of PBMCs on H929 (BCMA knockout) cells mediated by the exemplary antibody-3. [Figure 3G] Figure 3G shows the lethal effect of PBMCs on H929 cells mediated by the exemplary antibody-1. [Figure 3H] Figure 3H shows the toxic effect of PBMCs on H929 cells mediated by the exemplary antibody-2. [Figure 3I] Figure 3I shows the lethal effect of PBMCs on H929 cells mediated by the exemplary antibody-3. [Figure 3J] Figure 3J shows the lethal effect of PBMCs on L363 cells mediated by the exemplary antibody-1. [Figure 3K] Figure 3K shows the toxic effect of PBMCs on L363 cells mediated by the exemplary antibody-2. [Figure 3L] Figure 3L shows the toxic effect of PBMCs on L363 cells mediated by the exemplary antibody-3. [Figure 3M] Figure 3M shows the lethal effect of PBMCs on L363 cells mediated by the exemplary antibody-4. [Figure 3N] Figure 3N shows the toxic effect of PBMCs on mixed cells mediated by the exemplary antibody-1. [Figure 3O] Figure 3O shows the toxic effect of PBMCs on mixed cells mediated by the exemplary antibody-2. [Figure 3P] Figure 3P shows the toxic effect of PBMCs on non-target cells Calu-6 mediated by the exemplary antibody. [Figure 3Q] Figure 3Q shows the release levels of three types of cytokines (under three antibody concentration conditions) released during the H929-killing process of PBMCs mediated by the exemplary antibody. [Figure 3R] Figure 3R shows the release levels of three cytokines (under three antibody concentration conditions) released during the H929-killing process of PBMCs mediated by the exemplary antibody. [Figure 3S]Figure 3S shows the release of non-antigen-specific cytokines from PBMCs mediated by the exemplary antibody in the absence of target cells - 1 (under 3 antibody concentration conditions). [Figure 3T] Figure 3T shows the release of non-antigen-specific cytokines from PBMCs mediated by the exemplary antibody in the absence of target cells - 2 (under 3 antibody concentration conditions). [Figure 3U] Figure 3U shows the catastrophic effect of exemplary antibodies on PBMCs against GS CHO cells overexpressing BCMA mutants. [Figure 4A] Figure 4A shows the activation of CD4 T cells in PBMCs mediated by exemplary antibodies in the presence of soluble BCMA (sBCMA). [Figure 4B] Figure 4B shows the activation of CD8 T cells in PBMCs mediated by exemplary antibodies in the presence of soluble BCMA (sBCMA). [Figure 4C] Figure 4C shows the mediated PBMC killing of H929 cells by exemplary antibodies in the presence of soluble BCMA (sBCMA). [Figure 5A] Figure 5A shows the tumor-suppressing effect of exemplary antibodies in a humanized mouse model carrying H929 (GPRC5D knockout) tumors. Dosage and statistical methods are shown in the legend in the figure. Administration was performed on days 7, 14, and 21 after tumor cell inoculation. [Figure 5B] Figure 5B shows the tumor-suppressing effect of exemplary antibodies in a humanized mouse model carrying H929 (BCMA knockout) tumors. Dosage and statistical methods are shown in the legend in the figure. Administration was performed on days 7 and 14 after tumor cell inoculation. [Figure 5C] Figure 5C shows the tumor-suppressing effect of exemplary antibodies in a humanized mouse model carrying MM1S tumors. Dosages are indicated in the legend in the figure, and administration is performed on days 7 and 14 after tumor cell inoculation. [Figure 5D] Figure 5D shows the tumor-suppressing effect of the exemplary antibody in a humanized mouse model carrying the L363 tumor, with administration performed on days 7 and 18 after tumor cell inoculation. [Figure 6]Figure 6 shows the expression results of BCMA and GPRC5D in H929, H929 (GPRC5D knockout), and H929 (BCMA knockout) cell lines, as determined by flow cytometry. [Figure 7] Figure 7 shows the difference in aggregation between the B3(24) molecule and the control molecule (F2(24)). [Modes for carrying out the invention]

[0025] Unless otherwise defined, all technical and scientific terms used herein have the meaning generally understood by those skilled in the art. All publications, patent applications, patents, and other documents referenced herein are incorporated herein by whole-word by reference. Furthermore, the materials, methods, and examples described herein are illustrative examples only and are not intended to be limiting. Other features, purposes, and advantages of the present invention will become apparent from the specification and drawings and the appended claims.

[0026] definition The present invention is not limited to the specific methodologies, protocols, and reagents described herein, and these may be modified. Furthermore, the terms used herein are for the purpose of describing specific embodiments and do not limit the scope of the invention. The scope of the invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art.

[0027] For the purposes of this specification, the following definitions are used. Where necessary, a singular term includes a plural form, and vice versa.

[0028] When used in combination with a number, the term "approximately" is intended to encompass numbers that fall within a range from -5% to +5% of the given number.

[0029] As used herein, the terms "and / or" mean one, more than one, or all of the options.

[0030] As used herein, “comprise” or “include” means to include the elements, integers, or steps described, but not to exclude other elements, integers, or steps. Unless otherwise specified herein, “comprise” or “include” also includes cases where the whole consists of the elements, integers, or steps described. For example, when referring to an antibody variable region that “includes” a particular sequence, it is intended to include the antibody variable region consisting of that particular sequence.

[0031] The term "GPRC5D" refers to a tumor-associated antigen, G protein-coupled receptor family C group 5 member D (e.g., the human GPRC5D protein with UniProt accession number Q9NZD1). In one embodiment, the antigen-binding domain of the antibody molecule of the present invention that binds to GPRC5D has high affinity binding activity to GPRC5D-expressing cells, and may have an EC50 value of 1 to 150 nM, for example 20 to 135 nM, for human GPRC5D-expressing cells, as measured, for example by flow cytometry. In one embodiment, this antigen-binding specificity exhibits cross-reactivity to human and monkey GPRC5D.

[0032] The term "BCMA" refers to the tumor-associated B-cell maturation antigen (also known as BCMA, TR17_HUMAN, TNFRSF17; for example, the human BCMA protein with UniProt accession number Q02223). "BCMA" includes not only the wild-type protein but also mutants, such as naturally occurring mutants and mutants mutated in disease (particularly mutants with mutations that induce drug resistance). In one embodiment, the antigen-binding domain of the antibody molecule of the present invention that binds to BCMA has high affinity binding activity to BCMA, and is measured, for example, by biolayer interferometry (BLI), to human BCMA with K DThe value may be 0.1 to 10 nM, for example, 0.125 nM. In one embodiment, the antigen-binding specificity exhibits cross-reactivity to human and monkey BCMAs. The term "CD3" refers to the T cell engager T cell surface glycoprotein CD3 (e.g., human CD3 protein with UniProt accession number P07766). In one embodiment, the antigen-binding region of the antibody molecule of the present invention that binds to CD3 has high affinity binding activity to CD3 (e.g., CD3E and CD3G), and the KD value for human CD3E and CD3G may be 10 to 100 nM, for example, 10 to 70 nM, as measured by biolayer interferometry. In one embodiment, this antigen-binding specificity exhibits cross-reactivity to human and monkey CD3.

[0033] In this specification, references to "the first," "the second," and "the third" are used solely to distinguish between three domains or three chains, or three antigen-binding regions, and do not in any sense indicate the location of the three domains or three antigen-binding regions.

[0034] In describing the antibody structure of the present invention, the term "N-terminus / N-terminal side" refers to the final amino acid at the N-terminus, and the term "C-terminus / C-terminal side" refers to the final amino acid at the C-terminus.

[0035] As used herein, the terms “binding” or “specific binding” mean that the binding effect 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 immunosorbent assay (ELISA) or conventional binding assays known in the art.

[0036] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X to its counterpart molecule Y is generally expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (kdis and kon, respectively). Affinity can be measured by common methods known in the art. One specific method for affinity measurement described herein is the ForteBio kinetic binding assay.

[0037] The term "target" refers to the substance to which a binding molecule is directed. The target may be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production, activation of specific immune cells, or both. Those skilled in the art will understand that virtually any macromolecule, including all proteins or peptides, can be used as an antigen. Furthermore, the antigen may be derived from recombinant DNA or genomic DNA. As used herein, the term "epitope" refers to a part of an antigen that specifically interacts with an antibody molecule. In some embodiments, the antigen is a tumor-associated antigen (i.e., an antigen associated with the development and progression of a tumor) or a T-cell engager.

[0038] As used herein, the term “target-binding region” refers to a portion of a multispecific binding molecule (e.g., a triplicate binding molecule) that binds to a specific target or antigen. The target-binding region may be, for example, the antibody or immunoglobulin itself, or an antibody fragment. Such a target-binding region may or may not have a tertiary structure independent of the rest of the multispecific antibody molecule, and may or may not bind to a target as an independent unit. The target-binding region may be a receptor or ligand, or a receptor domain capable of binding to a ligand. In the case of a multispecific antibody, the “target-binding region” is also referred to as the “antigen-binding region.” In one embodiment, the antigen-binding region used in the multispecific antibody molecule of the present invention includes a VH / VL pair consisting of a light chain variable region (VL) and a heavy chain variable region (VH) of the antibody, and the VH / VL pair may be encapsulated in two separate polypeptide chains (e.g., contained in a Fab heavy chain and a Fab light chain, respectively). In one embodiment, the antigen-binding region used in the triplicate antibody molecule of the present invention may be Fab.

[0039] As used herein, "IgG-like multispecific antibody" refers to a multispecific antibody containing an Fc dimer.

[0040] The term "multispecificity binding molecule" refers to a multispecificity binding molecule that is at least bispecific (e.g., triplicate), that is, the molecule comprises at least a first target binding region, a second target binding region, and a third target binding region, where the first target binding region binds to one target, the second target binding region binds to another target, and the third target binding region binds to yet another third target. Thus, the multispecificity binding molecule of the present invention has specificity for at least two different targets. In some embodiments, when the binding molecule is an antibody, the target is an antigen. In some embodiments, the multispecificity binding molecule of the present invention is a multispecific antibody, for example, a triplicate antibody.

[0041] As used herein, a “multispecific” antibody refers to an antibody having at least two antigen-binding regions, where each antigen-binding site binds to a different epitope of the same antigen or to a different epitope of different antigens. A multispecific antibody is an antibody that has binding specificity to at least two different antigen epitopes. In one embodiment, this specification provides a tripspecific antibody having binding specificity to a first antigen, a second antigen, and a third antigen. For example, the present invention provides a tripspecific antibody targeting BCMA, GPRC5D, and CD3.

[0042] The reference to the "first antigen-binding region" in multispecific or trispecific antibodies refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody, for example, so that the multispecific antibody may contain one or more first antigen-binding regions. For example, a trispecific antibody contains a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region, but may contain one or more first antigen-binding regions, one or more second antigen-binding regions, or one or more third antigen-binding regions. In some embodiments, the antibody molecule of the present invention includes at least one antigen-binding region that specifically binds to GPRC5D, at least one antigen-binding region that specifically binds to BCMA, and at least one antigen-binding region that specifically binds to CD3. In some embodiments, the antibody molecule of the present invention includes one antigen-binding region that specifically binds to GPRC5D, one antigen-binding region that specifically binds to BCMA, and one antigen-binding region that specifically binds to CD3.

[0043] The reference to "antibody-derived antigen-binding region" means that the binding domain constituting the antigen-binding region is either the binding domain of the antibody that specifically binds to the antigen, or is derived from it. This includes cases where, for example, a fragment (Fab, etc.) of the antigen-binding region that specifically binds to the antigen is either the corresponding fragment (Fab, etc.) of the antibody, or is derived from it; or where the heavy chain variable region and / or light chain variable region of the antigen-binding region is either the heavy chain variable region and / or light chain variable region of the antibody, or is derived from them; or where one, two, three, four, five, or six CDRs of the antigen-binding region are CDRs of the antibody. "Derived from" means that a fragment in the antigen-binding region is substantially identical to the fragment of the antibody from which it is derived, but has one or more mutations such as substitutions, deletions, or additions. In certain embodiments, such mutations are not present in the antibody's CDRs. In certain embodiments, such mutations are not present in the antibody's variable regions.

[0044] In this specification, the terms "full-length antibody" and "full-length antibody" are used synonymously and refer to antibody molecules having the structure of a natural immunoglobulin molecule. In the case of conventional quadruple-chain IgG antibodies, the full-length antibody contains two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of heavy-chain antibodies that lack light chains and have only heavy chains, the full-length antibody contains two heavy chains (H) linked together by disulfide bonds. In conventional quadruple-chain IgG antibodies, the heavy chain of the full-length antibody generally consists of a heavy chain variable region (abbreviated as VH in this specification) and a heavy chain constant region, and the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of the full-length antibody consists of a light chain variable region (abbreviated as VL in this specification) and a light chain constant region, and the light chain constant region consists of one domain CL. Each heavy chain variable region (VH) or each light chain variable region consists of three CDRs and four FRs (framework regions), arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminus to the C-terminus. The term "antibody fragment" encompasses a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0045] An "antigen-binding fragment" of an antibody is a molecule distinct from the full-length antibody, containing a portion of the full-length antibody, but capable of binding to the antigen of the full-length antibody, or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for binding to the antigen. Antigen-binding fragments may be prepared by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv(scFv), diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by papain digestion of a full-length antibody. Furthermore, F(ab')2, a dimer of Fab', is a bivalent antibody fragment obtained by pepsin digestion of the area below the disulfide bond (C-terminal side) in the hinge region of a complete antibody. F(ab')2 can be converted to a Fab' monomer by cleaving the disulfide bond in the hinge region and reducing it under neutral conditions. The Fab' monomer is essentially a Fab fragment with a hinge region. The Fv fragment consists of a VL domain and a VH domain derived from one arm of the antibody. The two domains, VL and VH, of the Fv fragment may be encoded by separate genes, but they may also be linked together with a synthetic linker peptide using recombination techniques and expressed as a single protein chain, where VL and VH pair up to form a single-chain Fv (scFv).

[0046] The "variable region" refers to the heavy or light chain domains of an antibody that are involved in binding to an antigen. The variable regions of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). In some cases, antigen-binding specificity can be conferred by a single VH domain or VL domain alone.

[0047] In this specification, "Fab fragment" and "Fab" are used synonymously and refer to an immunoglobulin fragment consisting of two polypeptide chains, comprising an immunoglobulin heavy chain variable region VH, a heavy chain constant domain CH1, a light chain variable region VL, and a light chain constant domain CL, where one polypeptide chain contains one constant region selected from VH, CH1, and CL from the N-terminus to the C-terminus, and the other polypeptide chain contains one other constant region selected from VL, CL, and CH1 from the N-terminus to the C-terminus, with the VH and VL domains forming a pair to form an antigen-binding site. In this specification, the polypeptide chain of Fab containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain," and correspondingly, the polypeptide chain of Fab containing the light chain constant region CL is also referred to as the "Fab light chain."

[0048] The "complementarity-determining region," "CDR region," or "CDR" refers to a region in the variable region of an antibody that has a highly variable sequence, forms a structurally defined loop ("hypervariable loop"), and / or contains an antigen contact residue ("antigen contact site"). CDRs are primarily responsible for binding to the antigen epitope. CDRs in the heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. CDRs located in the variable region of the heavy chain of an antibody are referred to as HCDR1, HCDR2, and HCDR3, while CDRs located in the variable region of the light chain of an antibody are referred to as LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of the light chain variable region or heavy chain variable region, the precise amino acid sequence boundary of each CDR can be determined using one or a combination of well-known CDR assignment schemes, such as Chothia (Chothia et al., 1989, Nature 342:877-883; Al-Lazikani et al., 1997, J. Mol. Biol. 273:927-948) based on the three-dimensional structure of the antibody and the topology of the CDR loop; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., NIH, 1987) based on the variability of the antibody sequence; AbM (University of Bath); Contact (University College London); the international IMGT database (imgt.cines.fr on the World Wide Web); and the North CDR definition based on affinity propagation clustering using numerous crystal structures.

[0049] The terms “Fc domain” or “Fc region” are used herein to define a region of the C-terminal area of ​​an immunoglobulin heavy chain that includes at least a portion of the constant region. The “Fc region” includes the native sequence Fc region and mutant Fc regions. The “Fc domain” of a native immunoglobulin includes two or three constant domains, namely the CH2 domain, the CH3 domain, and optionally the CH4 domain. For example, in a native antibody, the Fc domain of the immunoglobulin 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. Unless otherwise specified herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU Index, for example, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can form a dimer Fc through dimerization, and two different Fc regions can form a heterodimer Fc through heterodimerization. In this specification, “Fc region,” “Fc portion,” and “dimer Fc (e.g., heterodimer Fc)” do not include the heavy chain variable region VH and light chain variable region VL, as well as the heavy chain constant region CH1 and light chain constant region CL of immunoglobulins, but may, in some cases, include the hinge region located at the N-terminus of the heavy chain constant region. In one embodiment, the Fc region of the human IgG heavy chain extends from Asp221, Cys226, or Asp231 to the C-terminus of the heavy chain.

[0050] In some embodiments, the multispecific antibody of the present invention includes an Fc region. In one embodiment, the Fc region is a human-derived Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass.

[0051] In one embodiment, the Fc region polypeptide (including the hinge region) of human IgG1 contains the following amino acid sequence.

[0052] [ka]

[0053] The Fc region polypeptide (including the hinge region) of human IgG4 contains the following amino acid sequence.

[0054] [ka]

[0055] In this specification, "heterodimeric Fc" refers to a scaffold consisting of two different Fc regions, or formed by the dimerization of two different Fc regions, which can be used to construct a multispecific antibody (e.g., a bispecific antibody) by linking an antigen-binding domain (e.g., a variable region of the heavy and / or light chain of an antibody, an antigen-binding fragment of an antibody that can bind to a target molecule, or a soluble portion of a ligand or receptor that can bind to a target molecule) to its N-terminus or C-terminus.

[0056] The "CH1 region" refers to the portion of the antibody heavy chain polypeptide corresponding to EU position 118 to EU position 220 in the EU numbering system. In one embodiment, the CH1 domain is

[0057] [ka]

[0058] It contains an amino acid sequence consisting of the following. In one embodiment, the internal disulfide bond can be removed by mutating the cysteine ​​in CH1 to a non-cysteine, or a new disulfide bond can be reconstructed by mutating the non-cysteine ​​to a cysteine.

[0059] The "CH2 region" refers to the portion of the antibody heavy chain polypeptide corresponding to EU position 231 to EU position 340 (EU numbering system).

[0060] In one embodiment, the CH2 domain is

[0061] [ka]

[0062] It contains an amino acid sequence consisting of the following:

[0063] The "CH3 region" refers to the portion of the antibody heavy chain polypeptide corresponding to EU position 341 to EU position 447. In one embodiment, the CH3 domain is

[0064] [ka]

[0065] It contains an amino acid sequence consisting of the following:

[0066] The term "hinge region" refers to the portion of the antibody heavy chain polypeptide that links to the CH1 and CH2 regions in the wild-type antibody heavy chain. For example, the hinge region of IgG1 containing the sequences D221 to P230 in the EU numbering system is an example. Hinge regions of other IgG subclasses can be identified by alignment with cysteine ​​residues present in the hinge region of the IgG1 subclass sequence. The hinge region is generally a dimeric molecule consisting of two polypeptides having the same amino acid sequence. In one embodiment, the hinge region has the amino acid sequence DKTHTCPXCP (SEQ ID NO: 99), where X is S or P. In one embodiment, the hinge region includes the amino acid sequence HTCPXCP (SEQ ID NO: 19), where X is S or P. In another embodiment, the hinge region includes the amino acid sequence CPXCP (SEQ ID NO: 81), where X is S or P. In one embodiment, the internal disulfide bond of the hinge region is removed by mutating the cysteine ​​in the hinge region to a non-cysteine.

[0067] In this specification, the amino acid positions throughout the variable regions of the heavy and light chains are numbered according to the Kabat numbering system described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0068] In this specification, when referring to amino acid positions in antibody domains other than the variable region (e.g., constant regions such as the Fc region), they shall be numbered according to the EU numbering scheme described in Edelman, GM et al., Proc. Natl. Acad. USA, 63:78-85 (1969). Where a position number and / or amino acid residue is assigned to a particular antibody isotype, such assignment is intended to be applicable to the corresponding position and / or amino acid residue of any other antibody isotype, as is well known to those skilled in the art.

[0069] As used herein, "linker" refers to any molecule that enables the direct linking of different parts of a multispecific antibody. Examples of linkers that form covalent bonds between different parts of a multispecific antibody include, but are not limited to, peptide linkers and non-proteinoid polymers, which include polyethylene glycol (PEG), polypropylene glycol, polyalkylene oxide, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, the "peptide linker" according to the present invention refers to an amino acid sequence that links the amino acid sequences of each site of a multispecific antibody. Preferably, the peptide linker is long enough to link the two components while maintaining their respective conformations without inhibiting the desired activity. The peptide linker may, but may not, consist mainly of Gly, Ser, Ala, or Thr amino acid residues. Useful linkers include, for example, (GS) n (Sequence ID 112), (GSGGS) n (Sequence ID 113), (GGGGS) n (Sequence ID 114), (GGGS) n (Sequence ID 115), and (GGGGS) n This includes glycine-serine polymers such as G (SEQ ID NO: 116), where n is an integer of 1 or more (preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0070] "Isolated" antibodies refer to antibodies separated from components in the natural environment. In some embodiments, these antibodies are purified to a purity of over 95% or over 99% by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange, reverse-phase HPLC).

[0071] "Isolated" nucleic acids refer to nucleic acid molecules separated from components of the natural environment. Isolated nucleic acids include nucleic acid molecules that are normally present in the cell containing the nucleic acid molecule, but these nucleic acid molecules are located outside the chromosome or in a location different from their original location on the chromosome. "Isolated nucleic acids encoding antibodies" refer to one or more nucleic acid molecules encoding each chain or fragment of an antibody, including those present in a single vector, separated into different vectors, and present in one or more locations within a host cell.

[0072] The calculation of sequence identity between sequences is performed as follows.

[0073] To determine the identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimizing the comparison (for example, gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, and non-homologous regions may be excluded from the comparison). In a preferred embodiment, the aligned length of the reference sequence for comparison is at least 30%, preferably at least 40%, more preferably at least 50% or 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. Then, amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are compared. If a residue or base at a position in the first sequence is identical to one at the corresponding position in the second sequence, then the two sequences are identical at that position.

[0074] An "amino acid substitution" or "amino acid mutation" refers to replacing at least one amino acid residue in a given parent amino acid sequence with a different "substituting" amino acid residue. The substituted residue may be a "naturally occurring amino acid residue" (i.e., encoded by the genetic code) and may be selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Substitutions by one or more unnaturally occurring amino acid residues are also included in the definition of amino acid substitution as used herein. "Non-natural amino acid residues" refer to residues that, in addition to the naturally occurring amino acid residues mentioned above, can form covalent bonds with adjacent amino acid residues within a polypeptide chain. Examples of non-natural amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib, and other amino acid residue analogs.

[0075] "Conservative modification" or "conservative substitution" refers to an amino acid modification or alteration that does not significantly affect or change the binding properties of the antibody or antibody fragment containing the amino acid sequence in question. Such conservative modifications include conservative amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibody or antibody fragment of the present invention by standard methods well known to those skilled in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. A conservative substitution is an amino acid substitution in which an amino acid residue is replaced with an acid residue having a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with neutral polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0076] Amino acid mutations are expressed as "original amino acid, amino acid position, mutated amino acid". For example, if the mutation site is located in the V region, "Q39D" means that glutamine at position 39 in the Kabat numbering system is replaced by aspartic acid (D), and if the mutation site is located in the C region, "Q124C" means that glutamine at position 124 in the EU numbering system is replaced by cysteine ​​(C). When referring to an amino acid position in this invention, unless otherwise specified, it refers to the amino acid position based on the numbering of the IgG1 heavy chain or κ (kappa) light chain, that is, in addition to the position based on the numbering of the IgG1 heavy chain or κ light chain, it also includes the amino acid position on other heavy or light chains corresponding to that position. When describing a mutation, it should be noted that the original amino acid at a specific position may be the amino acid described, or it may be any other amino acid at that corresponding position.

[0077] In this specification, "knob-in-hole mutation" refers to introducing a mutation using the "knob-in-hole method" to form a protrusion (knob) at the interface of the first Fc polypeptide and a complementary cavity (hole) at the interface of the second Fc polypeptide. The "knob-in-hole method" is known in the art to be able to modify the interface between different chains of antibody molecules and promote the precise association of antibody molecule chains. Generally, this method introduces a "protrusion" at the interface of one chain and a corresponding "cavity" at the interface of the other paired chain so that the protrusion fits into the cavity. A preferred interface includes a CH3 domain derived from the heavy chain constant domain of one chain and a CH3 domain derived from the heavy chain constant domain of the other paired chain. The protrusion can be constructed by replacing a small side chain at the interface of the CH3 domain derived from the heavy chain constant domain of one chain with a larger side chain such as tyrosine or tryptophan. A corresponding cavity of similar size is constructed at the interface of the CH3 domain derived from the heavy chain constant domain of the other paired chain by substituting a larger side chain with a smaller side chain such as alanine or threonine. Alternatively, the interface consisting of the light chain CL domain and the heavy chain CH1 domain of the Fab fragment may be used to construct a protrusion-cavity interaction, thereby promoting precise heterodimerization between the two chains of the Fab fragment.

[0078] "Effector function" refers to the biological activity derived from the immunoglobulin Fc region, which varies depending on the immunoglobulin isotype. Examples of immunoglobulin effector functions include Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake in antigen-presenting cells, C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0079] Antibody-dependent cell-mediated cytotoxicity (ADCC) refers to one of the major mechanisms by which specific cytotoxic effector cells (e.g., natural killer (NK) cells) mediate the killing of target cells or foreign host cells. In some embodiments, the antibodies of the present invention provide antibody-dependent cytotoxicity to T lymphocytes and enhance antibody-dependent cytotoxicity to NK cells.

[0080] Antibody-dependent phagocytosis (ADCP) refers to a cellular response in which target cell-binding antibodies bind to FcγRIIIa on the surface of macrophages, activating them, which then engulf target cells and acidify and degrade them using phagosomes. ADCP can be mediated by FcγRIIa and FcγRI, but their proportion is relatively small.

[0081] "Pharmaceutical composition" means a composition in which the biological activity of the contained active ingredients is effectively expressed, and which does not contain any additional ingredients that have unacceptable toxicity to the subject to which the composition is administered.

[0082] "Pharmaceutical adjuvants" refer to pharmaceutically acceptable carriers, diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete forms)), excipients, buffers, stabilizers, etc., that are administered together with the active ingredient.

[0083] A "conjugate" is an antibody that has been bound to one or more other substances (including, but not limited to, therapeutic agents or labels).

[0084] The terms "individual" and "subject" are used synonymously and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, horses), primates (e.g., humans and non-human primates such as monkeys), rabbits and rodents (e.g., mice and rats), etc. In particular, the individual or subject in question is a human.

[0085] "Tumor" refers to all neoplastic cell proliferation and modes of proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. "Tumor" includes solid tumors and humoral tumors. In some embodiments, such tumor is cancer. "Cancer" and "cancerous" refer to physiological diseases in mammals in which cell proliferation becomes uncontrolled. In this specification, "cancer," "cancerous," and "tumor" are not mutually exclusive concepts.

[0086] As used herein, “treat” (or “treating”) means delaying, interrupting, stopping, reducing, inhibiting, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. Desired therapeutic effects include, but are not limited to, prevention of disease onset or recurrence, symptom reduction, reduction of direct or indirect pathological consequences of the disease, inhibition of metastasis, delay of disease progression, improvement or mitigation of the condition, and improvement or mitigation of the prognosis. In some embodiments, the antibody molecules of the present invention are used to delay or slow the progression of disease.

[0087] As used herein, “prevention” (or “preventing” or “preventing”) includes the prevention of a disease or disorder, or the suppression of the onset or progression of symptoms of a particular disease or disorder. In some embodiments, subjects with a family history are candidates for a preventive regimen. Generally, in the context of cancer, “prevention” refers to administering a drug to subjects at particular risk of cancer, before the onset of signs or symptoms of cancer.

[0088] "Effective dose" refers to the amount or dosage of the antibody or composition of the present invention that produces the expected effect after one or more doses in a patient requiring treatment or prevention.

[0089] The "therapeutic dose" refers to the amount that is effective in the required dose for the duration necessary to achieve the desired therapeutic effect. The therapeutic dose is also the amount in which the toxicity or undesirable effects of the antibody, antibody fragment, or composition thereof are less than the beneficial therapeutic effect.

[0090] The "prophylactic effective dose" refers to the amount of medication that is effective at the required dosage for the duration necessary to achieve the desired preventive effect. Generally, since prophylactic doses are administered to patients before the onset of disease or in the early stages, the prophylactic effective dose is less than the therapeutic effective dose.

[0091] "Pharmaceutical combination" refers to a combination product of non-fixed doses or fixed doses, including but not limited to kits. "Non-fixed dose combination" means administering active ingredients (e.g., (i) the antibody of the present invention, (ii) an additional therapeutic agent) to a patient simultaneously or sequentially (without specific time restrictions, at the same or different dosing intervals) as separate entities, thereby providing the patient with two or more prophylactically or therapeutically effective active ingredients. In some embodiments, the antibody of the present invention used in a pharmaceutical combination is administered at a level not exceeding the dose when used alone. "Fixed dose combination" means administering two or more active ingredients to a patient simultaneously in a single formulation. The doses and / or dosing intervals of the two or more active ingredients are preferably selected so that the combined use of each ingredient provides a therapeutic effect against the disease or disorder that is greater than that of using either ingredient alone. Each ingredient may take an independent formulation form, and these independent formulation forms may be the same or different.

[0092] "Combination therapy" refers to the administration of two or more therapeutic agents or modalities (e.g., radiotherapy, surgery) to treat the diseases described herein. Such administration includes, for example, substantially simultaneous combination administration in a single capsule containing the active ingredients in a fixed ratio. Alternatively, it includes combination administration of active ingredients contained in various or separate containers, such as tablets, capsules, powders, or liquids. Powders and / or liquids may be reconstituted or diluted to the desired dose before administration. Furthermore, it includes cases where the various therapeutic agents are used nearly simultaneously or sequentially at different times. In any case, the treatment regimen provides a beneficial combined effect in the treatment of the disorders or symptoms described herein.

[0093] As used herein, the term "vector" refers to a nucleic acid molecule capable of replicating other ligated nucleic acids. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors that are integrated into the genome of a host cell. Some vectors can direct the expression of operable ligated nucleic acids. Such vectors are referred to herein as "expression vectors."

[0094] "Host cell" refers to a cell into which an exogenous polynucleotide has been introduced, and includes the progeny cells of said cell. Host cells include "transformed organisms" and "transformed cells," which include primary transformed cells and their derived progeny cells, regardless of the number of passages. Host cells are any cell lineage capable of producing the antibody molecules of the present invention, and include eukaryotic cells (e.g., mammalian cells, insect cells, yeast cells) and prokaryotic cells (e.g., Escherichia coli). In addition to cultured cells, host cells include cells in transgenic animals or transgenic plants, or cells in cultured plant tissue or cultured animal tissue.

[0095] As used herein, "label" refers to a compound or composition that is directly or indirectly conjugated or fused to an agent such as an antibody, thereby facilitating the detection of the agent to which it is conjugated or fused. The label itself may be detectable (e.g., radioisotope labeling, fluorescent labeling), and in the case of enzymatic labeling, it may catalyze a chemical change to a detectable substrate compound or composition. The term encompasses both direct labeling, which involves coupling (i.e., physically linking) a detectable substance to a probe or antibody, and indirect labeling, which involves reacting the probe with another directly labeled reagent. Examples of indirect labeling include the detection of a primary antibody using a fluorescently labeled secondary antibody, and the end labeling of a biotinylated DNA probe to make it detectable with fluorescently labeled streptavidin.

[0096] "Subject / patient sample" refers to a collection of cells, tissues, or bodily fluids obtained from a patient or subject. Tissue or cell samples may originate from solid tissues (e.g., fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples), blood or any blood component, bodily fluids such as cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid, and cells derived from the subject during pregnancy or at any point in development. Tissue samples may contain compounds not normally present in tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics. Examples of tumor samples include, but are not limited to, tumor biopsies, fine-needle aspirations, bronchial lavage fluid, pleural fluid, sputum, urine, surgically resected specimens, circulating tumor cells, serum, plasma, circulating plasma proteins, ascites, primary cultured cells or cell lines of tumor origin or tumor-like properties, and preserved tumor samples such as formalin-fixed paraffin-embedded (FFPE) tumor samples or frozen tumor samples.

[0097] As used herein, "detection" includes quantitative or qualitative detection. Typical detection methods include, but are not limited to, immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads combined with antibody molecules, ELISA assays, and PCR methods (e.g., RT-PCR). In some embodiments, the biological sample includes body fluids, cells, or tissues. In certain embodiments, the biological sample is blood, serum, or other biologically derived liquid samples.

[0098] All documents, patent applications, patents, and other reference materials referenced herein are incorporated herein by reference in their entirety. Any features described above and throughout this application can be combined with each other in various embodiments. Furthermore, the materials, methods, and examples described herein are illustrative and not intended to be limiting. Other features, purposes, and advantages of the present invention will become apparent from this specification, the drawings, and the appended claims.

[0099] I. Multispecific antibodies One aspect of the present invention relates to a triple-specific antibody, such as a triple-specific antibody, comprising a first antigen-binding region that specifically binds to BCMA and second and third antigen-binding regions that specifically bind to the second and third antigens.

[0100] One aspect of the present invention relates to a triplicate antibody that specifically binds to GPRC5D, BCMA, and CD3, comprising a first antigen-binding region that specifically binds to BCMA, a second antigen-binding region that specifically binds to CD3, and a third antigen-binding region that specifically binds to GPRC5D.

[0101] In some embodiments, the first antigen-binding region is derived from an anti-BCMA antibody or its antigen-binding fragment (e.g., a Fab fragment of an anti-BCMA antibody). In some embodiments, the first antigen-binding region is derived from an anti-BCMA antibody or its antigen-binding fragment as described herein.

[0102] In some embodiments, the second antigen-binding region is derived from an anti-CD3 antibody or its antigen-binding fragment (e.g., a Fab fragment of an anti-CD3 antibody). In some embodiments, the second antigen-binding region is derived from an anti-CD3 antibody or its antigen-binding fragment as described herein.

[0103] In some embodiments, the third antigen-binding region is derived from an anti-GPRC5D antibody or its antigen-binding fragment (e.g., a Fab fragment of an anti-GPRC5D antibody). In some embodiments, the third antigen-binding region is derived from an anti-GPRC5D antibody or its antigen-binding fragment as described herein.

[0104] The first antigen-binding region used in the triplicate antibody of the present invention may include or consist of the full-length anti-BCMA antibody or its antigen-binding fragment, as long as it can specifically bind to BCMA. Examples include, but are not limited to, full-length antibodies, single-chain Fv(scFv), Fab, Fab', (Fab)2, single-domain antibodies, VHH, heavy-chain antibodies, etc. Preferably, the first antigen-binding region is a Fab that specifically binds to BCMA.

[0105] The second antigen-binding region used in the triplicate antibody of the present invention may include or consist of a full-length anti-CD3 antibody or its antigen-binding fragment, as long as it can specifically bind to CD3. Examples include, but are not limited to, full-length antibodies, single-chain Fv(scFv), Fab, Fab', (Fab)2, single-domain antibodies, VHH, and heavy-chain antibodies. Preferably, the second antigen-binding region is a Fab that specifically binds to CD3.

[0106] The third antigen-binding region used in the triplicate antibody of the present invention may include or consist of a full-length anti-GPRC5D antibody or its antigen-binding fragment, as long as it can specifically bind to GPRC5D. Examples include, but are not limited to, full-length antibodies, single-chain Fv(scFv), Fab, Fab', (Fab)2, single-domain antibodies, VHH, and heavy-chain antibodies. Preferably, the third antigen-binding region is a Fab that specifically binds to GPRC5D.

[0107] In some embodiments, the triplicate antibody of the present invention is an IgG-like triplicate antibody. In some embodiments, the triplicate antibody of the present invention comprises an Fc dimer (e.g., a heterodimer Fc).

[0108] In some embodiments, the triplicate antibody of the present invention includes a Fab that specifically binds to BCMA as the first antigen-binding region.

[0109] In some embodiments, all three antigen-binding regions of the triplicate antibody of the present invention are Fab fragments.

[0110] Antigen-binding region suitable for use in the triplicate antibody of the present invention In some embodiments, the antigen-binding region of the triplicate antibody of the present invention is a Fab fragment.

[0111] A Fab fragment suitable for use in the antigen-binding region of the aforementioned triplicate antibody is composed of two polypeptide chains containing the VH, CH1, VL, and CL domains of the antibody, where VH is paired with VL and CH1 is paired with CL to form the antigen-binding region. In some embodiments, one chain of the Fab contains VH-CH1 from the N-terminus to the C-terminus, and the other chain contains VL-CL from the N-terminus to the C-terminus.

[0112] In some embodiments, in a triplicate antibody, Fab may be fused to the N-terminus of the Fc domain of the antibody via the C-terminus of the VH-containing chain, or fused to the C-terminus of the Fc domain via the N-terminus of the VH-containing chain. Here, the Fc domain may or may not include a hinge region. In some embodiments, the Fab consists of a VH-CH1 chain and a VL-CL chain, and is fused to the N-terminus of the Fc domain of the antibody via the C-terminus of CH1 of the VH-CH1 chain, or fused to the C-terminus of the Fc domain via the N-terminus of VH of the VH-CH1 chain.

[0113] In some embodiments, the fusion is direct fusion or linker-mediated fusion. In some embodiments, the linker is (GGGGS)n, where n is 1, 2, 3, or 4.

[0114] In some embodiments, the antigen-binding region, such as Fab, in the triplicate antibody of the present invention has been mutated to reduce mispairing.

[0115] In some embodiments, one antigen-binding region (e.g., Fab fragment) of the triplicate antibody of the present invention contains a disulfide bond remodeling mutation.

[0116] In some embodiments, one antigen-binding region of the triplicate antibody of the present invention (e.g., Fab fragment) includes a charge mutation in which each amino acid in an amino acid pair is replaced with an amino acid with an opposite charge.

[0117] In some embodiments, one antigen-binding region (e.g., Fab fragment) of the triplicate antibody of the present invention includes a charge mutation and a disulfide bond remodeling mutation.

[0118] In some embodiments, of the two antigen-binding regions (e.g., two Fab fragments) of the triplicate antibody of the present invention, one contains a charge mutation, and the other contains a charge mutation and a disulfide bond remodeling mutation. In some embodiments, the two antigen-binding regions each contain a charge mutation at a corresponding position. Preferably, the two antigen-binding regions contain charge mutations that are opposite to each other.

[0119] Disulfide bond remodeling mutation In some embodiments, a disulfide bond remodeling mutation refers to a mutation in the antigen-binding region CH1-CL in which the heavy chain CH1 (e.g., IgG1, IgG2, IgG3, or IgG4) is replaced with a cysteine ​​residue at EU position 126, and the light chain CL is replaced with a cysteine ​​residue at EU position 124. In some embodiments, the remodeling mutation includes 126C of CH1 (e.g., F126C) and 124C of CL (e.g., Q124C) (both EU numbers). In some embodiments, the remodeling mutation further includes a mutation that replaces native cysteine ​​with non-cysteine. In some embodiments, the antigen-binding region CH1 is replaced with a cysteine ​​residue at EU position 220. In some embodiments, the light chain CL of the antigen-binding region is replaced with a cysteine ​​residue at EU position 214. In some embodiments, the remodeling mutations include C220S / A / V of CH1 (IgG1 subtype; corresponding position C131S / A / V in IgG2, IgG3, or IgG4) and C214S / A / V of CL (both EU numbers). In some embodiments, Fab includes CH1 and CL, with CH1 having the F126C mutation and CL having the Q124C mutation (EU number). In some embodiments, the antigen-binding region includes CH1 and CL, with CH1 having F126C and C220S (or corresponding position C131S) and CL having Q124C and C214S (EU number).

[0120] In some embodiments, the Fab fragment containing the disulfide bond remodeling mutation comprises CH1, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 85, and contains F126C. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85. In some embodiments, CH1 comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 86, and contains F126C and C220S. In some embodiments, CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 86.

[0121] In some embodiments, the Fab fragment containing a disulfide bond remodeling mutation includes CL, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 97, and includes Q124C. In some embodiments, CL includes or consists of the amino acid sequence shown in SEQ ID NO: 97. In some embodiments, CL includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 98, and includes Q124C and C124S. In some embodiments, CL includes or consists of the amino acid sequence shown in SEQ ID NO: 98.

[0122] Charge mutation: In some embodiments, a charge mutation is a mutation relating to an amino acid pair located at the interface between the heavy chain and light chain of at least one antigen-binding region, wherein two amino acids in the amino acid pair are replaced with amino acids having opposite charges. In some embodiments, the charge mutation includes a mutation in which, according to Kabat numbering, each amino acid in the amino acid pair located at position 39 of the heavy chain variable region and position 38 of the light chain variable region is replaced with an amino acid of opposite charge. In some embodiments, the charge mutation includes a mutation in which, according to Kabat numbering, each amino acid in the amino acid pair consisting of Q39 of the heavy chain variable region and Q38 of the light chain variable region is replaced with an amino acid of opposite charge.

[0123] In some embodiments, in one antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with K, and the amino acid at position 38 of the light chain variable region is substituted with D. In some embodiments, in one antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with D, and the amino acid at position 38 of the light chain variable region is substituted with K.

[0124] In this specification, "two antigen-binding regions containing oppositely charged mutations" means that the first antigen-binding region and the second antigen-binding region have a charge mutation at one or more of the same amino acid pair positions, but the charge mutation of the amino acid pair in one antigen-binding region is different from the charge mutation of the amino acid pair in the other antigen-binding region. For example, at the same position, the charge of the mutated amino acid in the first antigen-binding region is opposite to the charge of the mutated amino acid in the second antigen-binding region. For example, in the first antigen-binding region, the heavy chain amino acid at position X1 is replaced with a positively charged amino acid, and the light chain amino acid at position X2 is replaced with a negatively charged amino acid. Also, in the second antigen-binding region, the heavy chain amino acid at position X1 is replaced with a negatively charged amino acid, and the light chain amino acid at position X2 is replaced with a positively charged amino acid. In this case, the positively charged amino acid in the first antigen-binding region may be the same as or different from the positively charged amino acid in the second antigen-binding region (e.g., same), and the negatively charged amino acid may also be the same as or different from the positively charged amino acid in the second antigen-binding region (e.g., same).

[0125] In some embodiments, in the first antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a positively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a negatively charged amino acid; and / or in the second antigen-binding region, the amino acid at position 39 of the heavy chain variable region is substituted with a negatively charged amino acid, and the amino acid at position 38 of the light chain variable region is substituted with a positively charged amino acid.

[0126] In one embodiment, the triplicate antibody of the present invention comprises two antigen-binding regions, in which the amino acid at position 39 of the heavy chain variable region is replaced with K and the amino acid at position 38 of the light chain variable region is replaced with D in one antigen-binding region, and in the other antigen-binding region, the amino acid at position 39 of the heavy chain variable region is replaced with D and the amino acid at position 38 of the light chain variable region is replaced with K.

[0127] antigen binding region In some embodiments, the triplicate antibody of the present invention includes an antigen-binding region that specifically binds to BCMA, an antigen-binding region that specifically binds to CD3, and an antigen-binding region that specifically binds to GPRC5D.

[0128] Antigen-binding domain that specifically binds to BCMA In some embodiments, the antigen-binding region that specifically binds to BCMA includes three complementarity-determining regions derived from the heavy chain variable region (HCDR), namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions derived from the light chain variable region (LCDR), namely LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions derived from the heavy chain variable region (HCDR1 to HCDR3) and three complementarity-determining regions derived from the light chain variable region (LCDR1 to LCDR3).

[0129] In some embodiments, the antigen-binding region that specifically binds to BCMA includes a heavy chain variable region (VH). In some embodiments, the antigen-binding region that specifically binds to CD3 includes a light chain variable region (VL). In some embodiments, the antigen-binding region that specifically binds to BCMA includes both a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) consisting of HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) consisting of LCDR1, LCDR2, and LCDR3. In some embodiments, the HCDRs and LCDRs are identified by the Kabat numbering system (as described in Sequences of Proteins of Immunological Interest, 5th edition, NIH, 1991).

[0130] In some embodiments, the heavy chain variable region (VH) of the antigen-binding region that specifically binds to BCMA is: (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in Sequence ID No. 56, or consisting of the said sequence; or, (ii) Consists of or comprises the amino acid sequence shown in Sequence ID No. 56; or, (iii) An amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in Sequence ID No. 56, or consisting of the above sequence. In this case, it is preferable that such amino acid modifications do not occur in the CDR.

[0131] In some embodiments, the light chain variable region (VL) of the antigen-binding region that specifically binds to BCMA is: (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in Sequence ID No. 60, or consisting of the said sequence; or, (ii) containing the amino acid sequence shown in Sequence ID No. 60, or consisting of the said sequence; or, (iii) An amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in Sequence ID No. 60, or consisting of the above sequence. In this case, it is preferable that such amino acid modifications do not occur in the CDR.

[0132] In some embodiments, the three complementarity-determining regions (HCDR1, HCDR2, HCDR3) derived from the heavy chain variable region of the antigen-binding domain that specifically binds to BCMA are HCDR1, HCDR2, and HCDR3 contained in VH as described in Sequence ID No. 56.

[0133] In some embodiments, the three complementarity-determining regions (LCDR1, LCDR2, LCDR3) derived from the light chain variable region of the antigen-binding region that specifically binds to BCMA are LCDR1, LCDR2, and LCDR3 contained in the VL described in SEQ ID NO: 60.

[0134] In some embodiments, in the antigen-binding region that specifically binds to BCMA of the present invention, HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO: 57; HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO: 58; HCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO: 59; LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO: 61; LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO: 62; and / or LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO: 63.

[0135] In some specific embodiments, the antigen-binding region that specifically binds to BCMA comprises VH and VL, where VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 56, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to said sequence; VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to said sequence.

[0136] In some specific embodiments, the antigen-binding region that specifically binds to BCMA includes three complementarity-determining regions (HCDR1, HCDR2, HCDR3) contained in VH as described in SEQ ID NO: 56 and three complementarity-determining regions (LCDR1, LCDR2, LCDR3) contained in VL as described in SEQ ID NO: 60.

[0137] In some specific embodiments, the antigen-binding regions that specifically bind to BCMA include HCDR1 (SEQ ID NO: 57), HCDR2 (SEQ ID NO: 58), HCDR3 (SEQ ID NO: 59), LCDR1 (SEQ ID NO: 61), LCDR2 (SEQ ID NO: 62), and LCDR3 (SEQ ID NO: 63).

[0138] In some specific embodiments, the antigen-binding region that specifically binds to BCMA comprises VH and VL, where VH comprises the amino acid sequence shown in SEQ ID NO: 56 and VL comprises the amino acid sequence shown in SEQ ID NO: 60.

[0139] In one embodiment, the antigen-binding region that specifically binds to BCMA is an antigen-binding fragment of an anti-BCMA antibody, and is selected from Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody (e.g., VHH), domain antibody (dAb), and linear antibody. Preferably, the antigen-binding region that specifically binds to BCMA is Fab.

[0140] In some embodiments, the first antigen-binding region in the triplicate antibody of the present invention is a Fab fragment that specifically binds to BCMA. In some embodiments, the Fab fragment that specifically binds to BCMA in the triplicate antibody of the present invention is derived from an anti-BCMA antibody.

[0141] In some embodiments, the Fab fragments that specifically bind to BCMA and are suitable for use in the triplicate antibodies of the present invention include disulfide bond remodeling mutations, for example, CH1 containing F126C or F126C and C220S, and CL containing Q124C or Q124C and C214S.

[0142] In some embodiments, the Fab fragment that specifically binds to BCMA and is suitable for use in the triplicate antibody of the present invention comprises a charge mutation, for example, containing Q39K in the heavy chain variable region and Q38D in the light chain variable region; or containing Q38K in the light chain variable region and Q39D in the heavy chain variable region.

[0143] A Fab fragment that specifically binds to BCMA and is suitable for use in the triplicate antibody of the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, containing Q39K in the heavy chain variable region and F126C or F126C and C220S in CH1; further comprising Q38D in the light chain variable region and Q124C or Q124C and C214S in CL.

[0144] A Fab fragment that specifically binds to BCMA and is suitable for use in the triplicate antibody of the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, containing Q39D in the heavy chain variable region and F126C or F126C and C220S in CH1; further comprising Q38K in the light chain variable region and Q124C or Q124C and C214S in CL.

[0145] In some embodiments, the Fab that specifically binds to BCMA and is suitable for use in the triplicate antibody of the present invention comprises a Fab heavy chain including a heavy chain variable region and a Fab light chain including a light chain variable region, wherein the heavy chain variable region of the Fab heavy chain is (i) containing the amino acid sequence shown in Sequence ID No. 76, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 76, and containing the Q39K mutation; and / or, The variable region of the Fab light chain is (i) containing the amino acid sequence shown in Sequence ID No. 75, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 75, and containing the Q38D mutation.

[0146] Antigen-binding domain that specifically binds to GPRC5D In some embodiments, the antigen-binding region that specifically binds to GPRC5D includes three complementarity-determining regions (HCDR1, HCDR2, HCDR3) derived from the heavy chain variable region. In some embodiments, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions (LCDR1, LCDR2, LCDR3) derived from the light chain variable region. In some embodiments, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions (HCDR1-HCDR3) derived from the heavy chain variable region and three complementarity-determining regions (LCDR1-LCDR3) derived from the light chain variable region.

[0147] In some embodiments, the antigen-binding region that specifically binds to GPRC5D includes a heavy chain variable region (VH). In some embodiments, the antigen-binding region that specifically binds to GPRC5D includes a light chain variable region (VL). In some embodiments, the antigen-binding region that specifically binds to GPRC5D includes both a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) consisting of HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) consisting of LCDR1, LCDR2, and LCDR3. In some embodiments, the HCDRs and LCDRs are identified by the Kabat numbering scheme (the numbering scheme described in Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0148] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to GPRC5D, for example, from an antibody that specifically binds to GPRC5D disclosed in PCT / CN2022 / 076832. In some embodiments, the antigen-binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region comprises 1, 2, and 3 CDRs of the heavy chain variable region (i.e., HCDR1, HCDR2, HCDR3) of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region comprises 1, 2, and 3 CDRs of the light chain variable region (i.e., LCDR1, LCDR2, LCDR3) of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region comprises 3 CDRs of the heavy chain variable region and 3 CDRs of the light chain variable region of a known antibody that specifically binds to GPRC5D. In some embodiments, the antigen-binding region includes heavy chain and light chain variable regions of a known antibody that specifically binds to GPRC5D.

[0149] In some embodiments, the heavy chain variable region (VH) of the antigen-binding domain that specifically binds to GPRC5D is: (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 40 or 48, or consisting of the said sequence; or, (ii) comprising or consisting of the amino acid sequence shown in Sequence ID No. 40 or 48; or, (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in Sequence ID No. 40 or 48, or consisting of the above sequence. In this case, it is preferable that such amino acid modifications do not occur in the CDR.

[0150] In some embodiments, the light chain variable region (VL) of the antigen-binding region that specifically binds to GPRC5D is: (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 44 or 52, or consisting of the said sequence; or, (ii) comprising or consisting of the amino acid sequence shown in Sequence ID No. 44 or 52; or, (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1) amino acid modifications (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in Sequence ID No. 44 or 52, or consisting of the above sequence. In this case, it is preferable that such amino acid modifications do not occur in the CDR.

[0151] In some embodiments, the three complementarity-determining regions (HCDR1, HCDR2, HCDR3) derived from the heavy chain variable region of the antigen-binding domain that specifically binds to GPRC5D are HCDR1, HCDR2, and HCDR3 contained in the VH described in either SEQ ID NO: 40 or 48.

[0152] In some embodiments, the three complementarity-determining regions (LCDR1, LCDR2, LCDR3) derived from the light chain variable region of the antigen-binding region that specifically binds to GPRC5D are LCDR1, LCDR2, LCDR3 contained in the VL described in either SEQ ID NO: 44 or 52.

[0153] In some embodiments, in the antigen-binding region that specifically binds to GPRC5D of the present invention, HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO: 41; HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO: 42; HCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO: 43; LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO: 45; LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO: 46; and / or LCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO: 47; or HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 49; HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 50; HCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 51; LCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 53; LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 54; and / or LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 55.

[0154] In a particular embodiment of the present invention, the antigen-binding region that specifically binds to GPRC5D includes VH and VL, where, VH contains, or consists of, the amino acid sequence shown in SEQ ID NO: 40, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with said sequence, and VL contains, or consists of, the amino acid sequence shown in SEQ ID NO: 44, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with said sequence; or, VH includes, or consists of, the amino acid sequence shown in SEQ ID NO: 48, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with said sequence; and VL includes, or consists of, the amino acid sequence shown in SEQ ID NO: 52, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with said sequence.

[0155] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D includes three complementarity-determining regions (HCDR1, HCDR2, HCDR3) contained in the VH described in SEQ ID NO: 40 and three complementarity-determining regions (LCDR1, LCDR2, LCDR3) contained in the VL described in SEQ ID NO: 44. In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D includes three complementarity-determining regions (HCDR1, HCDR2, HCDR3) contained in the VH described in SEQ ID NO: 48 and three complementarity-determining regions (LCDR1, LCDR2, LCDR3) contained in the VL described in SEQ ID NO: 52.

[0156] In some specific embodiments of the present invention, the antigen-binding regions that specifically bind to GPRC5D include HCDR1 (SEQ ID NO: 41), HCDR2 (SEQ ID NO: 42), HCDR3 (SEQ ID NO: 43), LCDR1 (SEQ ID NO: 45), LCDR2 (SEQ ID NO: 46), and LCDR3 (SEQ ID NO: 47). In some specific embodiments of the present invention, the antigen-binding regions that specifically bind to GPRC5D include HCDR1 (SEQ ID NO: 49), HCDR2 (SEQ ID NO: 50), HCDR3 (SEQ ID NO: 51), LCDR1 (SEQ ID NO: 53), LCDR2 (SEQ ID NO: 54), and LCDR3 (SEQ ID NO: 55).

[0157] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises VH and VL, where VH comprises the amino acid sequence shown in SEQ ID NO: 40 and VL comprises the amino acid sequence shown in SEQ ID NO: 44.

[0158] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to GPRC5D comprises VH and VL, where VH comprises the amino acid sequence shown in SEQ ID NO: 48 and VL comprises the amino acid sequence shown in SEQ ID NO: 52.

[0159] In one embodiment, the antigen-binding region that specifically binds to GPRC5D is an antigen-binding fragment of an anti-GPRC5D antibody, and is selected from Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody (e.g., VHH), domain antibody (dAb), and linear antibody. Preferably, the antigen-binding region that specifically binds to GPRC5D is Fab.

[0160] In some embodiments, the third antigen-binding region in the triplicate antibody of the present invention is a Fab fragment that specifically binds to GPRC5D. In some embodiments, the Fab fragment that specifically binds to GPRC5D in the triplicate antibody of the present invention is derived from an anti-GPRC5D antibody.

[0161] In some embodiments, the Fab fragments that specifically bind to GPRC5D and are suitable for use in the triplicate antibody of the present invention include disulfide bond remodeling mutations, for example, CH1 containing F126C or F126C and C220S, and CL containing Q124C or Q124C and C214S.

[0162] In some embodiments, the Fab fragment that specifically binds to GPRC5D and is suitable for use in the triplicate antibody of the present invention comprises a charge mutation, for example, containing Q39K in the heavy chain variable region and Q38D in the light chain variable region; or containing Q38K in the light chain variable region and Q39D in the heavy chain variable region.

[0163] A Fab fragment that specifically binds to GPRC5D and is suitable for use in the triplicate antibody of the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, containing Q39K in the heavy chain variable region and F126C or F126C and C220S in CH1; further comprising Q38D in the light chain variable region and Q124C or Q124C and C214S in CL.

[0164] A Fab fragment that specifically binds to GPRC5D and is suitable for use in the triplicate antibody of the present invention comprises a disulfide bond remodeling mutation and a charge mutation, for example, containing Q39D in the heavy chain variable region and containing F126C or F126C and C220S in CH1; further, containing Q38K in the light chain variable region and containing Q124C or Q124C and C214S in CL.

[0165] In some embodiments, the Fab that specifically binds to GPRC5D and is suitable for use in the triplicate antibody of the present invention comprises a Fab heavy chain including a heavy chain variable region and a Fab light chain including a light chain variable region, wherein the heavy chain variable region of the Fab heavy chain is (i) containing the amino acid sequence shown in Sequence ID No. 80, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 80, and containing the Q39K mutation; and / or, The variable region of the Fab light chain is (i) containing the amino acid sequence shown in Sequence ID No. 79, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in Sequence ID No. 79, and containing the Q38D mutation.

[0166] Antigen-binding region that specifically binds to CD3 In some embodiments, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions (HCDR1, HCDR2, HCDR3) derived from the heavy chain variable region (VH). In some embodiments, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions (LCDR1, LCDR2, LCDR3) derived from the light chain variable region (VL). In some embodiments, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions (HCDR1-HCDR3) derived from the heavy chain variable region and three complementarity-determining regions (LCDR1-LCDR3) derived from the light chain variable region.

[0167] In some embodiments, the antigen-binding region that specifically binds to CD3 includes a heavy chain variable region (VH). In some embodiments, the antigen-binding region that specifically binds to CD3 includes a light chain variable region (VL). In some embodiments, the antigen-binding region that specifically binds to CD3 includes both a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) consisting of HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) consisting of LCDR1, LCDR2, and LCDR3. In some embodiments, the HCDRs and LCDRs are identified by the Kabat numbering system (the numbering system described in Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0168] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to CD3, for example, from the CD3 antibody disclosed in WO2022068809 (such as sp34.24 or sp34.87).

[0169] In some embodiments, the antigen-binding region contains 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD3 (e.g., sp34.24 or sp34.87 disclosed in WO2022068809).

[0170] In some embodiments, the antigen-binding region comprises one, two, and three heavy chain variable regions CDR (i.e., HCDR1, HCDR2, HCDR3) of a known antibody that specifically binds to CD3 (e.g., sp34.24 or sp34.87 disclosed in WO2022068809).

[0171] In some embodiments, the antigen-binding region comprises one, two, and three light chain variable regions CDRs (i.e., LCDR1, LCDR2, LCDR3) of a known antibody that specifically binds to CD3 (e.g., sp34.24 or sp34.87 disclosed in WO2022068809).

[0172] In some embodiments, the antigen-binding region comprises three heavy chain variable regions (CDR1-HCDR3) and three light chain variable regions (CDR1-LCDR3) of a known antibody that specifically binds to CD3 (e.g., sp34.24 or sp34.87 disclosed in WO2022068809).

[0173] In some embodiments, the antigen-binding region includes the heavy chain variable region (VH) and light chain variable region (VL) of a known antibody that specifically binds to CD3 (e.g., sp34.24 or sp34.87 disclosed in WO2022068809), as well as the mutations described herein.

[0174] In some embodiments, the heavy chain variable region (VH) of the antigen-binding region that specifically binds to CD3 is: (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 64 or 72, or consisting of the said sequence; or, (ii) comprising or consisting of the amino acid sequence shown in Sequence ID No. 64 or 72; or, (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence shown in Sequence ID No. 64 or 72, or consisting of the above sequence, preferably such modifications do not occur in the CDR.

[0175] In some embodiments, the light chain variable region (VL) of the antigen-binding region that specifically binds to CD3 is: (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in Sequence ID No. 68, or consisting of the said sequence; or, (ii) containing the amino acid sequence shown in Sequence ID No. 68, or consisting of the said sequence; or, (iii) an amino acid sequence having one or more (preferably 10 or fewer, more preferably 5, 4, 3, 2, or 1 or fewer) amino acid modifications (preferably amino acid substitutions, more preferably conservative substitutions) compared to the amino acid sequence shown in Sequence ID No. 68, or consisting of the above sequence, preferably such modifications do not occur in the CDR.

[0176] In some embodiments, the three complementarity-determining regions (HCDR1, HCDR2, HCDR3) derived from the heavy chain variable region of the antigen-binding region that specifically binds to CD3 are HCDR1, HCDR2, and HCDR3 contained in the VH described in either SEQ ID NO: 64 or 72.

[0177] In some embodiments, the three complementarity-determining regions (LCDR1, LCDR2, LCDR3) derived from the light chain variable region of the antigen-binding region that specifically binds to CD3 are LCDR1, LCDR2, and LCDR3 contained in the VL described in Sequence ID No. 68.

[0178] In some embodiments, in the antigen-binding region of the present invention that specifically binds to CD3, HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 65; HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 66; HCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 67 or 73; LCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 69; LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 70; and / or LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 71.

[0179] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 comprises VH and VL, where VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 64 or 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0180] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 includes three complementarity-determining regions, HCDR1, HCDR2, and HCDR3, contained in VH as described in SEQ ID NO: 64 or 72, and three complementarity-determining regions, LCDR1, LCDR2, and LCDR3, contained in VL as described in SEQ ID NO: 68.

[0181] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 includes HCDR1 as described in SEQ ID NO: 65, HCDR2 as described in SEQ ID NO: 66, HCDR3 as described in SEQ ID NO: 67 or 73, LCDR1 as described in SEQ ID NO: 69, LCDR2 as described in SEQ ID NO: 70, and LCDR3 as described in SEQ ID NO: 71.

[0182] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 comprises VH and VL, where VH comprises the amino acid sequence shown in SEQ ID NO: 64 or 72, and VL comprises the amino acid sequence shown in SEQ ID NO: 68.

[0183] In one embodiment, the antigen-binding region that specifically binds to CD3 is an antigen-binding fragment of an anti-CD3 antibody, and is selected from Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody (e.g., VHH), domain antibody (dAb), and linear antibody. Preferably, the antigen-binding region that specifically binds to CD3 is Fab.

[0184] In some embodiments, the second antigen-binding region in the triplicate antibody of the present invention is a Fab fragment that specifically binds to CD3. In some embodiments, the Fab fragment that specifically binds to CD3 in the triplicate antibody of the present invention is derived from an anti-CD3 antibody.

[0185] In some embodiments, the Fab fragments that specifically bind to CD3 and are suitable for use in the triplicate antibodies of the present invention include disulfide bond remodeling mutations, for example, CH1 containing F126C or F126C and C220S, and CL containing Q124C or Q124C and C214S.

[0186] In some embodiments, the Fab fragment that specifically binds to CD3 and is suitable for use in the triplicate antibody of the present invention comprises a charge mutation, for example, containing Q39K in the heavy chain variable region and Q38D in the light chain variable region; or containing Q38K in the light chain variable region and Q39D in the heavy chain variable region.

[0187] A Fab fragment that specifically binds to CD3 and is suitable for use in the triplicate antibody of the present invention comprises disulfide bond remodeling mutations and charge mutations, for example, Q39K in the heavy chain variable region, F126C in CH1, or F126C and C220S; further comprising Q38D in the light chain variable region, Q124C in CL, or Q124C and C214S.

[0188] A Fab fragment that specifically binds to CD3 and is suitable for use in the triplicate antibody of the present invention comprises disulfide bond remodeling mutations and charge mutations, for example, Q39D in the heavy chain variable region, F126C in CH1, or F126C and C220S; further comprising Q38K in the light chain variable region, Q124C in CL, or Q124C and C214S.

[0189] In some embodiments, the Fab that specifically binds to CD3 and is suitable for use in the triplicate antibody of the present invention comprises a Fab heavy chain including a heavy chain variable region and a Fab light chain including a light chain variable region, wherein the heavy chain variable region of the Fab heavy chain is (i) containing or consisting of the amino acid sequence shown in Sequence ID No. 77 or 78; or, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 77 or 78, and containing the Q39D mutation; and / or, The variable region of the Fab light chain is (i) containing or consisting of the amino acid sequence shown in Sequence ID No. 74; or, (ii) Having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 74, and containing an amino acid sequence that includes the Q38K mutation.

[0190] Fc dimers usable in the triplicate antibody of the present invention In one embodiment, the two Fc regions contained in the triplicate antibody of the present invention form an Fc dimer by dimerization. Preferably, the two Fc regions form a heterodimer Fc by heterodimerization.

[0191] In some embodiments, the first Fc region and the second Fc region may be different, and dimerization may form a scaffold of heterodimer Fc.

[0192] In some embodiments, the Fc region includes both naturally occurring Fc regions and mutant Fc regions. Naturally occurring Fc regions include the natural Fc sequences of various immunoglobulins, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., “IgG subclasses and allotypes: from structure to effector functions”, October 20, 2014, doi:10.3389 / fimmu.2014.00520). In some embodiments, the Fc region is human IgG Fc, such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region includes, or consists of, the amino acid sequence shown in SEQ ID NO: 87 or SEQ ID NO: 88, or an amino acid sequence having at least 90% identity to these (e.g., 95%, 96%, 97%, 99% or more identity).

[0193] In some embodiments, the Fc region of the present invention comprises CH2 and CH3 of the antibody. In some embodiments, the Fc region of the antibody may have an IgG hinge region or a portion thereof, for example, an IgG1 hinge region or a portion thereof, at its N-terminus. The hinge region may contain mutations.

[0194] As will be understood by those skilled in the art, in order to facilitate the formation of the multispecific antibody of the present invention as a heterodimer, the Fc region contained in the multispecific antibody of the present invention may contain mutations that promote heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced into the CH3 region of the two Fc regions.

[0195] Methods for promoting heterodimerization of the Fc region are known in the industry. For example, the CH3 region of the first Fc region and the CH3 region of the second Fc region are complementaryly modified so that each CH3 region (or the heavy chain containing it) does not homodimerize with itself, but heterodimerizes only with the other complementaryly modified CH3 region (therefore, the CH3 regions of the first and second Fc regions form heterodimers with each other, but homodimers are not formed between the CH3 regions of the first region and the CH3 regions of the second region). Preferably, based on Knob-in-Hole technology, corresponding Knob mutations and Hole mutations are introduced into the first Fc region and the second Fc region, respectively.

[0196] In certain embodiments, the CH3 region of one Fc region is modified by substituting the threonine residue at position 366 with a tryptophan residue (T366W) (knob mutation), while the CH3 region of the other Fc region is modified by substituting the tyrosine residue at position 407 with a valine residue (Y407V) (whole mutation). Optionally, the threonine residue at position 366 can be substituted with a serine residue (T366S) and / or the leucine residue at position 368 can be substituted with an alanine residue (L368A) (numbered according to the EU Index).

[0197] In yet another embodiment, in the CH3 region of one Fc region, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), and the serine residue at position 354 is replaced with a cysteine ​​residue (S354C) or the glutamic acid residue at position 356 is replaced with a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced with a cysteine ​​residue). On the other hand, in the CH3 region of the other Fc region, the tyrosine residue at position 407 is replaced with a valine residue (Y407V) (whole mutation), and optionally, the threonine residue at position 366 can be replaced with a serine residue (T366S) and the leucine residue at position 368 can be replaced with an alanine residue (L368A) (numbering according to EU number). Furthermore, the tyrosine residue at position 349 can be optionally replaced with a cysteine ​​residue (Y349C) (numbered according to the EU number).

[0198] In a particular embodiment, one Fc region contains the T366W mutation, and the other Fc region contains the T366S, L368A, and Y407V mutations (numbered according to EU numbering).

[0199] In a particular embodiment, one Fc region contains the S354C and T366W mutations, while the other Fc region contains the Y349C, T366S, L368A, and Y407V mutations (numbered according to EU numbering).

[0200] Therefore, in certain embodiments, the triplicate antibody of the present invention comprises two heterodimerized Fc regions, where one Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 91, and the other Fc region polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 92. Accordingly, in certain embodiments, the triplicate antibody of the present invention comprises two heterodimerized Fc regions, where one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 92.

[0201] Accordingly, in certain embodiments, the triplicate antibody of the present invention comprises two heterodimerized Fc regions, where one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 92 and includes the T366W mutation, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91 and includes the T366S, L368A, and Y407V mutations. In some embodiments, the Fc region further includes additional mutations that facilitate the purification of the heterodimer.

[0202] Mutations may be introduced into the binding molecule of the present invention (such as the Fc region of an antibody) to obtain desired properties. Mutations related to the Fc region are well known to those skilled in the art.

[0203] In one embodiment, the Fc region has modified characteristics of its effector function (e.g., complement activation function of the Fc region). In one embodiment, the effector function is reduced or absent compared to the Fc region of the wild-type isotype. In one embodiment, the effector function is reduced or absent by a method selected from using an Fc isotype in which the effector function is naturally reduced or absent, and by performing Fc region modification.

[0204] In a preferred embodiment, the Fc region has reduced or absent effector functions mediated by the Fc region (e.g., effector functions of ADCC / ADCP / CDC), and includes, for example, mutations to achieve the above functions.

[0205] As will be obvious to those skilled in the art, the binding molecule of the present invention (e.g., an antibody molecule) may further include modifications of the Fc domain to alter the binding affinity to one or more Fc receptors, depending on its desired application. In one embodiment, the Fc receptor is an Fcγ receptor, and in particular a human Fcγ receptor. In some embodiments, the Fc region includes mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has an L234A / L235A mutation that reduces binding to the Fcγ receptor. In a more preferred embodiment, the Fc fragment may have a mutation that extends its half-life in the blood, for example, a mutation that improves the binding of the Fc fragment to FcRn.

[0206] In some embodiments, the Fc region containing a mutation that reduces binding to the Fcγ receptor includes, or consists of, the amino acid sequence shown in SEQ ID NO: 89 or SEQ ID NO: 90, or an amino acid sequence having at least 90% (e.g., 95%, 96%, 97%, 99% or more) identity with them, or consists of the amino acid sequence.

[0207] In some embodiments, the Fc region includes an amino acid sequence having at least 90% (e.g., 95%, 96%, 97%, 99% or more) identity with the amino acid sequence shown in SEQ ID NO: 89 or SEQ ID NO: 90, or consists of the above amino acid sequence and includes the L234A / L235A mutation.

[0208] In a preferred embodiment, the heterodimer Fc used in the triplicate antibody of the present invention comprises two Fc regions, where, One Fc region contains or consists of the amino acid sequence shown in SEQ ID NO: 93, and the other Fc region contains or consists of the amino acid sequence shown in SEQ ID NO: 94; or One Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 94, or consists of the aforementioned amino acid sequence and includes the L234A / L235A and T366W mutations. The other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 93, or consists of the aforementioned amino acid sequence and includes the L234A / L235A, T366S, L368A, and Y407V mutations.

[0209] Structure of a triplicate antibody and examples of the triplicate antibody of the present invention In some embodiments, the present invention provides a triplicate antibody comprising a first Fab that specifically binds to BCMA, a Fab that specifically binds to CD3, a Fab that specifically binds to GPRC5D, and an Fc heterodimer.

[0210] In some embodiments, the present invention provides a triplicate antibody comprising a first Fab that specifically binds to BCMA, a Fab that specifically binds to CD3, a Fab that specifically binds to GPRC5D, and an Fc heterodimer.

[0211] In a preferred embodiment, the triplicate antibody of the present invention comprises a first Fab that specifically binds to a first antigen, a second Fab that specifically binds to a second antigen, a third Fab that specifically binds to a third antigen, and an Fc heterodimer, where the first antigen is selected from BCMA, CD3, and GPRC5D, the second antigen is selected from BCMA, CD3, and GPRC5D, the third antigen is selected from BCMA, CD3, and GPRC5D, and the first to third antigens are distinct from each other. (1) The first Fab that specifically binds to the first antigen is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions constituting the Fc heterodimer (the first Fc region, for example, the Fc region containing the Knob mutation or the Fc region containing the Hole mutation); (2) The second Fab that specifically binds to the second antigen is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of the other Fc region of the Fc heterodimer (the second Fc region; for example, the Fc region containing the Hole mutation, or the Fc region containing the Knob mutation); and (3) The third Fab that specifically binds to the third antigen is fused at the C-terminus of CH1 of the Fab heavy chain to the N-terminus of VH of the Fab heavy chain of the second Fab; or, The third Fab, which specifically binds to the third antigen, is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions constituting the Fc heterodimer (the first Fc region, for example, the Fc region containing the Knob mutation or the Fc region containing the Hole mutation); The second Fab, which specifically binds to the second antigen, is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of the other Fc region of the Fc heterodimer (the second Fc region; for example, the Fc region containing the Hole mutation, or the Fc region containing the Knob mutation); and The first Fab, which specifically binds to the first antigen, is fused to the N-terminus of the VH of the Fab heavy chain of the second Fab at the C-terminus of the CH1 of the Fab heavy chain.

[0212] For example, the structure is as shown in Figure 1A.

[0213] (4) The first Fab that specifically binds to the first antigen is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions constituting the Fc heterodimer (the first Fc region, for example, the Fc region containing the Knob mutation or the Fc region containing the Hole mutation).

[0214] The second Fab, which specifically binds to the second antigen, is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of the other Fc region of the Fc heterodimer (the second Fc region; for example, the Fc region containing the Hole mutation, or the Fc region containing the Knob mutation); and The third Fab, which specifically binds to the third antigen, is fused to the C-terminus of the Fc region fused to the second Fab at the N-terminus of the VH of the Fab heavy chain; or, The third Fab, which specifically binds to the third antigen, is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of one of the Fc regions constituting the Fc heterodimer (the first Fc region, for example, the Fc region containing the Knob mutation or the Fc region containing the Hole mutation).

[0215] The second Fab, which specifically binds to the second antigen, is fused at the C-terminus of CH1 of the Fab heavy chain to the CH2 or hinge region of the other Fc region of the Fc heterodimer (the second Fc region; for example, the Fc region containing the Hole mutation, or the Fc region containing the Knob mutation); and The first Fab, which specifically binds to the first antigen, is fused to the C-terminus of the Fc region fused to the second Fab at the N-terminus of the VH of the Fab heavy chain.

[0216] For example, the structure is as shown in Figure 1B.

[0217] In some embodiments, the second antigen is CD3. In some embodiments, the first and third antigens are GPRC5D or BCMA, respectively, or the third and first antigens are GPRC5D or BCMA, respectively.

[0218] In some embodiments, the first antigen is BCMA, the second antigen is CD3, and the third antigen is GPRC5D, or the first antigen is GPRC5D, the second antigen is CD3, and the third antigen is BCMA.

[0219] In some embodiments, the fusion includes direct fusion or fusion via a linker. In some embodiments, the linker is (GGGGS)n, where n = 1, 2, 3, or 4.

[0220] In some embodiments, the first Fc region includes a Knob mutation and the second Fc region includes a Hole mutation. In some embodiments, the first Fc region includes a Hole mutation and the second Fc region includes a Knob mutation. In some embodiments, the first and / or second Fc regions include an L234A / L235A mutation. In some embodiments, the first Fc region includes a Knob mutation and an L234A / L235A mutation, and the second Fc region includes a Hole mutation and an L234A / L235A mutation. In some embodiments, the first Fc region includes a Hole mutation and an L234A / L235A mutation, and the second Fc region includes a Knob mutation and an L234A / L235A mutation.

[0221] In some embodiments, the second Fab includes a charge mutation. In some embodiments, one or both of the first or third Fab include a charge mutation and a disulfide bond remodeling mutation.

[0222] In some embodiments, the second Fab contains a charge mutation, and either the first Fab or the third Fab contains a charge mutation and a disulfide bond remodeling mutation. In some embodiments, the two Fabs containing charge mutations contain opposing charge mutations.

[0223] In some embodiments, the second Fab includes a charge mutation, which includes the Q38K mutation in the Fab light chain variable region and the Q39D mutation in the Fab heavy chain variable region. In some embodiments, the first or third Fab includes a charge mutation and a disulfide bond remodeling mutation, which includes the Q39K mutation in the Fab heavy chain variable region and the F126C mutation in CH1, as well as the Q38D mutation in the Fab light chain variable region and the Q124C mutation in CL. In some embodiments, the first or third Fab includes a charge mutation and a disulfide bond remodeling mutation, which includes the Q39K mutation in the Fab heavy chain variable region, the F126C mutation and the C220S mutation in CH1, as well as the Q38D mutation in the Fab light chain variable region, the Q124C mutation and the C214S mutation in CL.

[0224] In some embodiments, the second Fab includes a charge mutation, which includes the Q38D mutation in the Fab light chain variable region and the Q39K mutation in the Fab heavy chain variable region. In some embodiments, the first or third Fab includes a charge mutation and a disulfide bond remodeling mutation, which includes the Q39D mutation in the Fab heavy chain variable region and the F126C mutation in CH1, as well as the Q38K mutation in the Fab light chain variable region and the Q124C mutation in CL. In some embodiments, the first or third Fab includes a charge mutation and a disulfide bond remodeling mutation, which includes the Q39D mutation in the Fab heavy chain variable region, the F126C mutation and the C220S mutation in CH1, as well as the Q38K mutation in the Fab light chain variable region, the Q124C mutation and the C214S mutation in CL.

[0225] In some embodiments, a first Fab or a third Fab comprising a charge variant and a disulfide bond remodeling variant is fused to a second Fab or fused to the second Fab via an Fc region.

[0226] In some specific embodiments, the trispecific antibody of the present invention is a left-right asymmetric IgG-like pentamer composed of five polypeptide chains, consisting of five polypeptide chains and comprising the following polypeptide chains.

[0227] Peptide chain 1# having in sequence a Fab heavy chain and an Fc domain that specifically binds to GPRC5D, Peptide chain 2# comprising a Fab light chain that specifically binds to GPRC5D, Peptide chain 3# having in sequence a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain, Peptide chain 4# comprising a Fab light chain that specifically binds to BCMA, Peptide chain 5# comprising a Fab light chain that specifically binds to CD3.

[0228] Here, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form a third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5# respectively to form a first Fab and a second Fab (shown in FIG. 1A as an example).

[0229] In some specific embodiments, peptide chain 1# comprises, or consists of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 5.

[0230] Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4.

[0231] Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 3 or 6, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3 or 6.

[0232] Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0233] Peptide chain 5# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0234] In some specific embodiments, peptide chain 1# includes, or consists of, the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 15.

[0235] Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 14.

[0236] Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 3 or 6, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3 or 6.

[0237] Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0238] Peptide chain 5# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0239] In some specific embodiments, the triplicate antibody of the present invention is a left-right asymmetric IgG-like pentamer consisting of five polypeptide chains, comprising the following peptide chains:

[0240] Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to BCMA, Peptide chain 2# containing a Fab heavy chain that specifically binds to BCMA, A Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and a peptide chain 3# having Fc domains in sequence, Peptide chain 4# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain #5 containing a Fab light chain that specifically binds to CD3.

[0241] Here, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the first Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the third Fab and second Fab (as shown in Figure 1A for example).

[0242] In some specific embodiments, peptide chain 1# includes, or comprises, the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11.

[0243] Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10.

[0244] Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 9.

[0245] Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 8.

[0246] Peptide chain 5# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0247] In some specific embodiments, the trispecific antibody of the present invention is a left-right asymmetric IgG-like pentamer composed of five polypeptide chains, consisting of five polypeptide chains and comprising the following polypeptide chains.

[0248] Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically binds to GPRC5D in sequence, Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain 3# having a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain of an antigen-binding region that specifically binds to BCMA in sequence, Peptide chain 4# containing a Fab light chain that specifically binds to CD3, Peptide chain 5# containing a Fab light chain of an antigen-binding site that specifically binds to BCMA.

[0249] Here, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form a third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5# respectively to form a second Fab and a first Fab (shown in Figure 1B as an example).

[0250] In some specific embodiments, peptide chain 1# comprises, or consists of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 5.

[0251] Peptide chain 2# comprises, or consists of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence shown in SEQ ID NO: 4.

[0252] Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7.

[0253] Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0254] Peptide chain 5# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0255] II. Production and Purification of Multispecific Antibodies In one embodiment, the present invention provides a method for producing an antibody molecule or a fragment thereof or a chain thereof. The method comprises culturing host cells under conditions suitable for expressing a nucleic acid encoding the antibody molecule or a fragment thereof or a chain thereof, and optionally further comprising separating the antibody or the fragment or the chain. In one embodiment, the method further comprises recovering the antibody molecule or a fragment thereof or a chain thereof from the host cells.

[0256] The polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Methods well known to those skilled in the art can be used to construct the expression vector. After an expression vector containing one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. For this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral introduction, viral infection, bioristics, liposomes, and other conventionally known methods can be used.

[0257] Antibody molecules prepared by the methods described herein can be purified by known techniques such as high-performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, and size exclusion chromatography (SEC). The actual conditions used for purifying specific proteins also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art. The purity of the antibody molecules of the present invention can be determined by any of the various well-known analytical methods such as size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0258] III. Immunoconjugates In some embodiments, the present invention provides an immunoconjugate comprising one of the antibodies provided herein and other substances such as a therapeutic agent or label. In some embodiments, the therapeutic agent may be a therapeutic agent suitable for forming an immunoconjugate with the antibody. In some embodiments, the therapeutic agent is a therapeutic agent selected from any of the following categories (i) to (iii): (i) an agent that enhances antigen presentation (e.g., tumor antigen presentation); (ii) an agent that enhances effector cell response (e.g., activation and / or recruitment of B cells and / or T cells); (iii) an agent that reduces immunosuppression; and (iv) an agent having antitumor activity. In some embodiments, the therapeutic agent is a chemotherapeutic agent.

[0259] In some embodiments, the immune conjugate is an antibody-drug conjugate (ADC).

[0260] IV. Nucleic acids and host cells The present invention provides nucleic acids encoding any chain, any monomer, any domain, or any antigen-binding site of the multispecific antibody (e.g., tripspecific antibody) of the present invention. The polynucleotide sequences encoding each chain can be prepared by methods well known to those skilled in the art. Furthermore, the polynucleotides and nucleic acids of the present invention include segments encoding secretion signal peptides and are operably linked to an antibody or protein encoding the multispecific antibody (e.g., tripspecific antibody) of the present invention, thereby inducing secretory expression of the multispecific antibody (e.g., tripspecific antibody) and each of its chains.

[0261] The present invention also provides vectors comprising nucleic acids disclosed herein. In one embodiment, the vector is an expression vector, for example, a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, cosmids, λ phages, or yeast artificial chromosomes (YACs). In a preferred embodiment, the expression vector of the present invention is a pCNDA vector, for example, a pCDNA3.1 expression vector.

[0262] The present invention further provides host cells containing the nucleic acid or the vector. Host cells suitable for supporting the replication and expression of the multispecific antibodies (e.g., tripspecific antibodies) of the present invention are well known to those skilled in the art. Such cells can be transfected or transduced with a specific expression vector, and vector-containing cells can be cultured in large quantities and inoculated into large-scale fermenters to obtain multispecific antibodies (e.g., tripspecific antibodies) sufficient for clinical application.

[0263] In one embodiment, the host cell is a eukaryote. In another embodiment, the host cell is selected from yeast cells and mammalian cells (e.g., CHO cells or 293 cells [such as Expi293 cells or HEK293 cells]), and examples of available mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7), human fetal kidney line (293 or 293T cells; e.g., Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells; e.g., Mather, Biol Reprod 23, Examples of available mammalian host cell lines include 243-251 (1980), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HeLa), canine kidney cells (MDCK), gerbil hepatocytes (BRL3A), human lung cells (W138), human hepatocytes (HepG2), mouse mammary cancer cells (MMT060562), TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383, 44-68 (1982)), MRC5 cells, and FS4 cells. Other available mammalian host cell lines include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells; Urlaub et al., Proc. Natl. Acad. Sci. USA 77, 4216 (1980)) and myeloma cell lines such as YO, NS0, P3X63, and Sp2 / 0. In one embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell such as Chinese hamster ovary (CHO) cells, human fetal kidney (HEK) cells, or lymphocytes (e.g., Y0, NS0, Sp20 cells).

[0264] V. Pharmaceutical compositions, pharmaceutical combinations, and kits In one embodiment, the present invention provides a composition, pharmaceutical, or formulation (e.g., a pharmaceutical composition) comprising the tripspecific antibody molecule of the present invention, optionally including a pharmaceutical auxiliary material. In some embodiments, the pharmaceutical auxiliary material is a pharmaceutically acceptable carrier.

[0265] The compositions of the present invention may take on a variety of forms. These forms include, for example, liquid, semi-solid, and solid formulations (liquid solutions [such as injectable and intravenous solutions], dispersions or suspensions, liposomes, and suppositories). The preferred form depends on the intended route of administration and therapeutic use. Generally, the preferred compositional form is an injectable or intravenous solution. The preferred form of administration is parenteral injection or intravenous infusion. As used herein, "parenteral administration" and "administered parenterally" mean a form of administration other than enteral and topical administration, and typically refers to administration by injection, including, but not limited to, intravenous, intramuscular, intra-arterial, intradermal, intraperitoneal, intratracheal, subcutaneous injection and intravenous infusion.

[0266] In a preferred embodiment, the antibody molecule is administered by intravenous infusion or intravenous injection. In another preferred embodiment, the antibody molecule is administered by intramuscular, intraperitoneal, or subcutaneous injection.

[0267] In some embodiments, the antibody molecule of the present invention is the sole active ingredient in the pharmaceutical composition. In other embodiments, the pharmaceutical composition may contain the antibody molecule described herein and further include one or more additional therapeutic agents.

[0268] The pharmaceutical composition of the present invention may contain a "therapeutic effective amount" or a "preventive effective amount" of the antibody molecule of the present invention.

[0269] Kits containing the antibody molecules described herein are also within the scope of the present invention. Such kits may include, but are not limited to, one or more other elements, such as a package insert, other reagents such as labeling and binding reagents, a pharmaceutically acceptable carrier, an apparatus for administration to a subject, or other materials.

[0270] In another embodiment, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the antibody molecule described herein and one or more additional therapeutic agents.

[0271] The present invention further provides a kit comprising the aforementioned pharmaceutical combination, for example, the kit comprising the following in the same packaging:

[0272] - A first container for containing a pharmaceutical composition comprising the triplicate antibody of the present invention; and - A second container for containing a pharmaceutical composition including additional therapeutic agents.

[0273] Additional therapeutic agents suitable for use in the pharmaceutical compositions and pharmaceutical combinations of the present invention may be selected from the following groups: (i) agents that enhance antigen presentation (e.g., tumor antigen presentation); (ii) agents that enhance effector cell responses (e.g., activation and / or recruitment of B cells and / or T cells); (iii) agents that reduce immunosuppression; and (iv) agents that have antitumor effects. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent.

[0274] VI. Uses and Methods of the Molecules of the Present Invention In one embodiment, the present invention provides in vivo and in vitro applications and methods of use of the antibody molecule of the present invention.

[0275] In some embodiments, the uses and methods of the present invention include applying the antibody molecule in vivo and / or in vitro to the following: - High affinity binding to GPRC5D antigen (including GPRC5D antigen expressed on the cell surface); - 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; - Activation of the CD3 downstream signaling pathway in T cells; - Mediating the killing effect of T cells against GPRC5D-positive tumor cells and / or BCMA-positive tumor cells; - Induction of cytokine release from T cells, such as TNF-α, IFN-γ, and IL-2; - Suppression or killing of GPRC5D-positive and / or BCMA-positive tumor cells; or - Treatment of GPRC5D-positive and / or BCMA-positive tumors such as multiple myeloma, e.g., treatment of BCMA-positive patient populations, GPRC5D-positive patient populations, or GPRC5D-positive patient populations in which BCMA has been eliminated by binding of an anti-BCMA molecule; or - Avoidance of tumor recurrence (e.g., recurrence of multiple myeloma) caused by BCMA escape.

[0276] In some embodiments, the present invention provides a method for preventing or treating GPRC5D and / or BCMA-related diseases in a subject, the method comprising administering to the subject a tripspecific antibody of the present invention, or an immunoconjugate, composition, pharmaceutical, or formulation containing said tripspecific antibody. In some embodiments, the present invention provides a method of use in a subject for the above-mentioned uses, the method comprising administering to the subject a tripspecific antibody of the present invention, or an immunoconjugate, composition, pharmaceutical, or formulation containing said tripspecific antibody.

[0277] In some embodiments, the disease is, for example, a tumor, i.e., cancer. The cancer may be in the early, intermediate, or advanced stage, or it may be metastatic cancer. In some embodiments, the cancer is a GPRC5D monopositive cancer, a BCMA monopositive cancer, a GPRC5D / BCMA bipositive cancer, or a bipolar low-expression cancer of both BCMA and GPRC5D (e.g., cancer that recurs after anti-BCMA therapy or anti-GPRC5D therapy). In some embodiments, the cancer is a solid tumor or a hematological malignancy. In some embodiments, the cancer is multiple myeloma.

[0278] By possessing antigen-binding specificity for BCMA and GPRC5D, the triplicate antibody molecule of the present invention may have a broader scope of application in the patient population for cancer treatment compared to BCMA / CD3-targeted bispecific antibodies and GPRC5D / CD3-targeted bispecific antibodies. In some embodiments, the present invention provides applications for the triplicate antibody molecule of the present invention in the treatment of GPRC5D monopositive cancer. In some embodiments, the present invention provides applications for the triplicate antibody molecule of the present invention in the treatment of BCMA monopositive cancer. In some embodiments, the present invention provides applications for the triplicate antibody molecule of the present invention in the treatment of GPRC5D / BCMA bipositive cancer. In some embodiments, the present invention further provides a method of treating cancers with low expression of BCMA and / or GPRC5D, such as GPRC5D low expression and / or BCMA low expression, or bi-low expression of BCMA and GPRC5D (e.g., cancer that recurs after anti-BCMA therapy and / or anti-GPRC5D therapy), using the triplicate antibody molecule of the present invention.

[0279] In this specification, "GPRC5D monopositive cancer" refers to cases where only GPRC5D expression is observed in the cancer cells, or where only GPRC5D expression is elevated (i.e., overexpressed) compared to healthy cells (e.g., cells of the same tissue in a healthy subject, or healthy cells adjacent to the cancer cells). In this specification, "GPRC5D low-expression cancer" refers to cases where the amount of GPRC5D expression in the cancer cells is low, for example, where it is expressed only slightly higher than in healthy cells of normal tissue.

[0280] In this specification, "BCMA monopositive cancer" refers to cases where only BCMA expression is observed in the cancer cells, or where only BCMA expression is elevated (i.e., overexpressed) compared to healthy cells (e.g., cells of the same tissue in a healthy subject, or healthy cells adjacent to the cancer cells). In this specification, "BCMA low-expression cancer" refers to cases where the amount of BCMA expression in the cancer cells is low, for example, where it is expressed only slightly higher than that of healthy cells in normal tissue.

[0281] In this specification, "GPRC5D / BCMA double-positive cancer" refers to cases in which GPRC5D expression and BCMA expression are observed in the cancer cells, or where the expression of GPRC5D and BCMA is elevated compared to healthy cells (e.g., cells from the same tissue of a healthy subject, or healthy cells adjacent to the cancer cells). "GPRC5D / BCMA double-positive cancer" also includes cancers that have BCMA expression and GPRC5D expression, but in which one or both are low in expression. For example, it includes cancers with low BCMA expression and / or low GPRC5D expression, or cancers with double low expression of BCMA and GPRC5D (e.g., cancers that recur after anti-BCMA therapy and / or anti-GPRC5D therapy).

[0282] In this specification, "BCMA-positive" includes not only cases where wild-type BCMA is expressed, but also cases where BCMA variants are expressed. BCMA variants may be variants obtained due to mutations such as deletions or amino acid substitutions. In some embodiments, BCMA mutations cause drug resistance to BCMA-targeted immunotherapy in individuals. Therefore, "BCMA-positive cancer" in this invention also includes cancers in which BCMA variant expression is observed, or cancers in which the expression of such BCMA variant is elevated (i.e., overexpressed) compared to healthy cells (e.g., cells of the same tissue in a healthy subject or healthy cells adjacent to the cancer cells).

[0283] In some embodiments, the BCMA variant includes one or more or all of the following mutations:

[0284] (i) Deletion of proline (P) at position 34 (P34del); (ii) Deletion of serine (S) at position 30 (S30del); (iii) amino acid substitution at position 39 (e.g., substitution of arginine (R) to alanine (A) (R39A)); and (iv) Amino acid substitution at position 27 (e.g., substitution of arginine (R) to proline (P) (R27P)).

[0285] In some embodiments, the present invention provides a method for suppressing or killing GPRC5D-positive and / or BCMA-positive cells.

[0286] In some embodiments, GPRC5D-positive cells are cells that express or overexpress GPRC5D within them.

[0287] In some embodiments, the BCMA variant in the BCMA-positive cells includes one or more or all of the following mutations:

[0288] (i) Deletion of proline (P) at position 34 (P34del); (ii) Deletion of serine (S) at position 30 (S30del); (iii) amino acid substitution at position 39 (e.g., substitution of arginine (R) to alanine (A) (R39A)); and (iv) Amino acid substitution at position 27 (e.g., substitution of arginine (R) to proline (P) (R27P)).

[0289] In some embodiments, BCMA-positive cells expressing or overexpressing the BCMA variant of the present invention are resistant to BCMA-targeted immunotherapy (e.g., erranatamab or teclistamadab treatment). In some embodiments, BCMA-positive cells expressing or overexpressing the BCMA variant of the present invention are resistant to BCMA and GPRC5D-targeted immunotherapy.

[0290] In some embodiments, BCMA-positive cells expressing or overexpressing the BCMA variant of the present invention have BCMA mutations of P34del, S30del, R39A, or R27P. In some embodiments, BCMA-positive cells expressing or overexpressing the BCMA variant of the present invention show expression of BCMA having mutations of P34del, S30del, R39A, or R27P, or such expression is elevated (i.e., overexpressed) compared to healthy cells (e.g., cells from the same tissue of a healthy subject or healthy cells adjacent to cancer cells).

[0291] In some embodiments, BCMA-positive cells expressing or overexpressing the BCMA variant of the present invention express or overexpress the BCMA variant containing a P34del or R27P mutation and exhibit resistance to erlanatamab.

[0292] In some embodiments, BCMA-positive cells expressing or overexpressing the BCMA variant of the present invention express or overexpress BCMA variants including P34del, S30del, R39A, or R27P mutations and exhibit resistance to tecristamag.

[0293] In some embodiments, the antibody molecule of the present invention, or a pharmaceutical composition containing the antibody molecule, is used as a pharmaceutical for the treatment and / or prevention of diseases associated with GPRC5D and / or BCMA, or as a pharmaceutical for killing or suppressing GPRC5D-positive and / or BCMA-positive cells in an individual, and as a diagnostic tool for GPRC5D and / or BCMA-related diseases, where the individual is preferably a mammal, more preferably a human.

[0294] In other embodiments, the present invention provides use of a triplicate antibody, or an immunoconjugate, composition, or combination formulation containing such antibody, in the uses described herein, for example, in the manufacture or preparation of a pharmacopoeci for the prevention or treatment of the related diseases or conditions referred to herein, or for the killing or suppression of GPRC5D-positive and / or BCMA-positive cells.

[0295] In some embodiments, the triplicate antibodies of the present invention (and immunoconjugates, compositions, pharmaceutical compositions, formulations, etc., containing such antibodies) may be administered in combination with one or more additional therapies (e.g., therapeutic modalities and / or additional therapeutic agents) for the uses described herein, for example, the prevention and / or treatment of the aforementioned related diseases or conditions, or for the killing or suppression of GPRC5D-positive and / or BCMA-positive cells.

[0296] In some embodiments, the treatment modality is, for example, surgery or radiotherapy.

[0297] 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., from a hyperproliferative or cancerous lesion), in vitro or in vivo. The diagnostic method comprises (i) contacting an antibody molecule described herein with a sample (optionally a control sample) or administering the antibody molecule to a target under conditions that allow for interaction, and (ii) detecting the formation of a complex between the antibody molecule and the sample (optionally a control sample). The formation of the complex indicates the presence of the relevant antigen and may indicate the suitability or necessity of the treatment and / or prophylaxis described herein.

[0298] In some embodiments, the associated antigens, such as GPRC5D and / or BCMA, are detected pre-treatment, for example, before the first treatment or before a predetermined treatment after a treatment interval. Possible 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 antibodies. Suitable detectable substances include various biologically active enzymes, prosthetic groups, fluorescent substances, luminescent substances, paramagnetic substances (e.g., nuclear magnetic resonance activators), and radioactive substances.

[0299] In some embodiments, the levels and / or distribution of related antigens such as GPRC5D and / or BCMA are measured in vivo. For example, the antibody molecule of the present invention is labeled with a detectable substance and detected non-invasively using appropriate imaging techniques such as positron emission tomography (PET) scanning. In one embodiment, for example, the levels and / or distribution of related antigens are measured in vivo by detecting the antibody molecule of the present invention, which has been detectably labeled with a PET reagent (e.g., 18F-fluorodeoxyglucose (FDG)).

[0300] In one embodiment, the present invention provides a diagnostic kit comprising the antibody molecule and accompanying documentation described herein.

[0301] VII. Specific Embodiments In one aspect of the present invention, the present invention relates to the following specific embodiments.

[0302] 1. A triplicate antibody that specifically binds to BCMA, comprising a first antigen-binding region that specifically binds to BCMA, and second and third antigen-binding regions that specifically bind to other antigens.

[0303] 2. The triplicate antibody according to Embodiment 1, wherein the second antigen-binding region specifically binds to CD3 and / or the third antigen-binding region specifically binds to GPRC5D.

[0304] 3. A triplicate antibody according to Embodiment 1 or 2, wherein the first antigen-binding region, the second antigen-binding region, and the third antigen-binding region are the first Fab, the second Fab, and the third Fab, respectively.

[0305] 4. A triplicate antibody according to Embodiment 3, wherein, (1) The first Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; and the third Fab fuses with the N-terminus of VH of the Fab heavy chain of the second Fab fragment at the C-terminus of CH1 of the Fab heavy chain; or The third Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; and the first Fab fuses with the N-terminus of VH of the Fab heavy chain of the second Fab fragment at the C-terminus of CH1 of the Fab heavy chain; or (2) The first Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, and the third Fab fuses with the C-terminus of the Fc region fused to the second Fab at the N-terminus of VH of the Fab heavy chain; or The third Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; and the first Fab fuses with the C-terminus of the Fc region fused to the second Fab at the N-terminus of VH of the Fab heavy chain; Trispecific antibodies.

[0306] 5. The triplicate antibody according to Embodiment 4, wherein the fusion includes direct fusion or linker-mediated fusion.

[0307] 6. A triplicate antibody according to Embodiment 5, characterized in that the linker is (GGGGS)n and n=1, 2, 3, or 4.

[0308] 7. A trispecific antibody according to any one of Embodiments 3 to 6, wherein the second Fab comprises a charge mutation, and the first or third Fab fused to the second Fab, or fused to the second Fab via an Fc region, comprises a charge mutation and a disulfide bond remodeling mutation.

[0309] 8. The triplicate antibody according to Embodiment 7, wherein the charge mutation is a mutation that replaces the 39th position of VH of Fab with D or K, and the 38th position of VL of Fab with K or D, for example, The second fabric includes VH containing 39D and VL containing 38K, and The first or third Fab, which is fused to the second Fab via the Fc region, includes VH containing 39K and VL containing 38D; or, The second fabric includes VH containing 39K and VL containing 38D, and The first or third Fab, which is fused to the second Fab via the Fc region, is a trispecific antibody containing VH with 39D and VL with 38K.

[0310] 9. A tripspecific antibody according to Embodiment 7 or 8, wherein the disulfide bond remodeling mutations include F126C in CH1 of Fab and Q124C in CL of Fab.

[0311] 10. The triplicate antibody according to Embodiment 9, wherein the Fab containing the disulfide bond remodeling mutation comprises CH1, where CH1 is (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 85, and containing F126C, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 85.

[0312] 11. A trispecific antibody according to Embodiment 9 or 10, wherein the Fab comprising a disulfide bond remodeling mutation includes CL, where CL is (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 97, and containing Q124C, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 97.

[0313] 12. A triplicate antibody according to Embodiment 9, wherein the Fab containing a disulfide bond remodeling mutation comprises CH1 and CL, where CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85, and CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 97.

[0314] 13. A tripspecific antibody according to Embodiment 7 or 8, wherein the disulfide bond remodeling mutations include F126C and C220S in CH1 of Fab and Q124C and C214S in CL of Fab.

[0315] 14. The triplicate antibody according to Embodiment 13, wherein the Fab containing the disulfide bond remodeling mutation comprises CH1, where CH1 is (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 86, and including F126C and C220S, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 86.

[0316] 15. A triplicate antibody according to Embodiment 13 or 14, wherein the Fab comprising a disulfide bond remodeling mutation includes CL, where CL is (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 98, and including Q124C and C214S, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 98.

[0317] 16. A triplicate antibody according to Embodiment 13, wherein the Fab containing a disulfide bond remodeling mutation comprises CH1 and CL, where CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 86, and CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 98.

[0318] 17. A triplicate antibody according to any one of Embodiments 1 to 16, wherein the triplicate antibody comprises a first Fc region and a second Fc region, wherein the first Fc region and the second Fc region are the same or different.

[0319] 18. A triplicate antibody according to Embodiment 17, wherein the first Fc region and the second Fc region are human IgG Fc, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, and for example, the antibody contains, or comprises, the amino acid sequence shown in SEQ ID NO: 87 or 88, or an amino acid sequence having at least 90% identity (e.g., 95%, 96%, 97%, 99% or more) to said sequence.

[0320] 19. A triplicate antibody according to Embodiment 18, wherein the first and / or second Fc region contains the L234A / L235A mutation.

[0321] 20. A triplicate antibody according to Embodiment 19, wherein the first and / or second Fc region comprises the amino acid sequence shown in SEQ ID NO: 89 or 90, or comprises an amino acid sequence having at least 90% identity (e.g., 95%, 96%, 97%, 99% or more) with respect to the amino acid sequence shown in SEQ ID NO: 89 or 90, and comprises the amino acid substitution L234A / L235A.

[0322] 21. A triplicate antibody according to any one of Embodiments 17 to 20, wherein one of the first Fc region and the second Fc region contains a Knob mutation and the other contains a Hole mutation.

[0323] 22. A triplicate antibody according to Embodiment 21, wherein one Fc region contains the amino acid substitution T366W, and the other Fc region contains the amino acid substitutions T366S, L368A, and Y407V (according to the EU numbering system).

[0324] 23. A triplicate antibody according to Embodiment 22, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 92.

[0325] 24. A triplicate antibody according to Embodiment 23, wherein one Fc region polypeptide contains or consists of the amino acid sequence shown in SEQ ID NO: 91, and the other Fc region polypeptide contains or consists of the amino acid sequence shown in SEQ ID NO: 92.

[0326] 25. A triplicate antibody according to Embodiment 17, wherein one Fc region comprises the amino acid substitutions L234A / L235A and T366W, and the other Fc region comprises the amino acid substitutions L234A / L235A, T366S, L368A and Y407V (according to EU numbering).

[0327] 26. A triplicate antibody according to Embodiment 25, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 94, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 93.

[0328] 27. A triplicate antibody according to Embodiment 26, wherein one Fc region polypeptide contains or consists of the amino acid sequence shown in SEQ ID NO: 94, and the other Fc region polypeptide contains or consists of the amino acid sequence shown in SEQ ID NO: 93.

[0329] 28. A trispecific antibody according to any one of Embodiments 1 to 27, wherein the first antigen-binding site that specifically binds to BCMA comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein A triple-specific antibody comprising the following characteristics: HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 57; HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 58; HCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 59; LCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 61; LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 62; and LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 63.

[0330] 29. A triplicate antibody according to Embodiment 28, wherein the VH of the first antigen-binding site that specifically binds to BCMA includes the amino acid sequence shown in SEQ ID NO: 56, or consists of the said sequence, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the said sequence.

[0331] 30. A triplicate antibody according to Embodiment 28 or 29, wherein the first antigen-binding site VL that specifically binds to BCMA includes, or comprises, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 60.

[0332] 31. A triplicate antibody according to Embodiment 30, wherein the first antigen-binding site that specifically binds to BCMA comprises VH and VL, where VH consists of the amino acid sequence shown in SEQ ID NO: 56, and the VL of the first antigen-binding site that specifically binds to BCMA consists of the amino acid sequence shown in SEQ ID NO: 60.

[0333] 32. A trispecific antibody according to Embodiment 28, wherein the first antigen-binding site that specifically binds to BCMA comprises VH and VL, wherein VH comprises 39K and VL comprises 38D, or, for example, VH of the first antigen-binding site comprises VH, (i) containing the amino acid sequence shown in Sequence ID No. 76, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 76, and including an amino acid sequence containing the Q39K mutation, and / or, the VL of the first antigen-binding site is (i) containing the amino acid sequence shown in Sequence ID No. 75, or consisting of the said sequence, (ii) A trispecific antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 75, and comprising an amino acid sequence containing the Q38D mutation.

[0334] 33. A trispecific antibody according to any one of Embodiments 1 to 32, wherein the third antigen-binding site specifically binds to GPRC5D and comprises a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3. HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 41; HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 42; HCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 43; LCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 45; LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 46; LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 47; or A triple-specific antibody comprising the following sequences: HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 49; HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 50; HCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 51; LCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 53; LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 54; and LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 55.

[0335] 34. The triplicate antibody according to Embodiment 33, wherein the VH of the third antigen-binding site that specifically binds to GPRC5D is (i) an amino acid sequence that includes, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in Sequence ID No. 40; or, (ii) A trispecific antibody comprising, or consisting of, the amino acid sequence shown in Sequence ID No. 48, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said sequence.

[0336] 35. A triplicate antibody according to Embodiment 33 or 34, wherein the VL of the third antigen-binding site that specifically binds to GPRC5D is (i) an amino acid sequence that includes, or consists of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in Sequence ID No. 44; or, (ii) A triplicate antibody comprising, or consisting of, the amino acid sequence shown in Sequence ID No. 52, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to said sequence.

[0337] 36. A triplicate antibody according to Embodiment 35, wherein the third antigen-binding site that specifically binds to GPRC5D comprises VH and VL, where VH consists of the amino acid sequence shown in SEQ ID NO: 48 and VL consists of the amino acid sequence shown in SEQ ID NO: 52; or VH consists of the amino acid sequence shown in SEQ ID NO: 40 and VL consists of the amino acid sequence shown in SEQ ID NO: 44.

[0338] 37. A trispecific antibody according to any one of embodiments 33 to 36, wherein the third antigen-binding site that specifically binds to GPRC5D comprises VH and VL, where VH comprises 39K and VL comprises 38D; or VH comprises 39D and VL comprises 38K. For example, the VH of the third antigen-binding site that specifically binds to GPRC5D is (i) containing the amino acid sequence shown in Sequence ID No. 80, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 80, and containing the Q39K mutation; And the VL of the third antigen-binding site is (i) containing the amino acid sequence shown in Sequence ID No. 79, or consisting of the sequence, (ii) A trispecific antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 79, and also comprising the Q38D mutation.

[0339] 38. A trispecific antibody according to any one of Embodiments 1 to 37, wherein the second antigen-binding site specifically binds to CD3 and comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein A triplicate antibody comprising the following characteristics: HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 65; HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 66; HCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 67 or SEQ ID NO: 73; LCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 69; LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 70; and LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 71.

[0340] 39. A triplicate antibody according to Embodiment 38, wherein the VH of the second antigen-binding site that specifically binds to CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 64 or SEQ ID NO: 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said sequence.

[0341] 40. A triplicate antibody according to any one of Embodiments 38 or 39, wherein the VL of the second antigen-binding site that specifically binds to CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 68, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said sequence.

[0342] 41. A triplicate antibody according to Embodiment 40, wherein the second antigen-binding site that specifically binds to CD3 comprises VH and VL, where VH consists of the amino acid sequence described in SEQ ID NO: 64 or SEQ ID NO: 72, and the VL of the second antigen-binding site consists of the amino acid sequence described in SEQ ID NO: 68.

[0343] 42. A triplicate antibody according to any one of embodiments 38 to 41, wherein the second antigen-binding site that specifically binds to CD3 comprises VH and VL, where VH comprises 39K and VL comprises 38D; or VH comprises 39D and VL comprises 38K.

[0344] 43. The triplicate antibody according to Embodiment 42, wherein the VH of the second antigen-binding site that specifically binds to CD3 is (i) containing the amino acid sequence described in Sequence ID No. 77 or Sequence ID No. 78, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence described in SEQ ID NO: 77 or 78, and containing the Q39D mutation; Furthermore, the VL of the second antigen-binding site that specifically binds to CD3 is (i) containing the amino acid sequence described in Sequence ID No. 74, or consisting of the said sequence, (ii) A trispecific antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 74, and comprising the Q38K mutation.

[0345] 44. A triplicate antibody according to any one of Embodiments 3 to 43, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer, consisting of five polypeptide chains, and comprising the following peptide chains: Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to GPRC5D.

[0346] Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D.

[0347] A peptide chain 3# having, in sequence, a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain.

[0348] Peptide chain 4# containing a Fab light chain that specifically binds to BCMA, and Peptide chain #5 containing a Fab light chain that specifically binds to CD3.

[0349] Here, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form a third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form a first Fab and a second Fab, respectively, in a triple-specific antibody.

[0350] 45. A triplicate antibody according to Embodiment 44, wherein, Peptide chain 1# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4; Peptide chain 3# contains, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 3 or 6; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2; and / or A triplicate antibody comprising peptide chain 5# containing, or consisting of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0351] 46. ​​A triplicate antibody according to Embodiment 44, wherein, Peptide chain 1# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 15; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 14; Peptide chain 3# contains, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 3 or 6; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2; and / or, A triplicate antibody comprising peptide chain 5# containing, or consisting of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0352] 47. A triplicate antibody according to any one of Embodiments 1 to 43, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer, consisting of five polypeptide chains, and comprising the following peptide chains Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to BCMA, Peptide chain 2# containing a Fab heavy chain that specifically binds to BCMA, A Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and a peptide chain 3# having Fc domains in sequence, Peptide chain 4# containing a Fab light chain that specifically binds to GPRC5D, and Peptide chain 5# containing a Fab light chain that specifically binds to CD3 A triplicate antibody consisting of In this antibody, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the first Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the third Fab and second Fab, respectively, resulting in a triple-specific antibody.

[0353] 48. A trispecific antibody as described in Embodiment 47, wherein, Peptide chain 1# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10; Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 9; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 8; and / or A triplicate antibody comprising peptide chain 5# containing, or consisting of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0354] 49. A triplicate antibody according to any one of Embodiments 1 to 43, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer, consisting of five polypeptide chains, the following peptide chains: Peptide chain 1# having a Fab heavy chain and Fc domain that specifically bind to GPRC5D. Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain 3# having sequentially a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain with an antigen-binding site that specifically binds to BCMA. Peptide chain 4# containing a Fab light chain that specifically binds to CD3, and Peptide chain 5# containing the Fab light chain, which is an antigen-binding site that specifically binds to BCMA. It consists of, In this triple-specific antibody, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the second Fab and first Fab, respectively.

[0355] 50. A triplicate antibody according to Embodiment 49, wherein, Peptide chain 1# contains, or consists of, an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4; Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1; and / or Peptide chain 5# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0356] 51. A nucleic acid molecule encoding any of the chains of the trispecific antibody described in any one of Embodiments 1 to 50, or a nucleic acid molecule consisting of a nucleic acid sequence.

[0357] 52. An expression vector comprising a nucleic acid molecule as described in Embodiment 51, wherein the expression vector is, for example, a pCNDA vector (e.g., a pcDNA3.1 expression vector).

[0358] 53. A host cell comprising the nucleic acid molecule described in Embodiment 51 or the expression vector described in Embodiment 52, wherein the host cell is preferably a prokaryotic or eukaryotic cell, for example, a 293 cell or a CHO cell (e.g., a HEK293 cell).

[0359] 54. A method for producing a tripspecific antibody according to any one of Embodiments 1 to 50, the method comprising culturing host cells containing a nucleic acid molecule according to Embodiment 51 or an expression vector according to Embodiment 52 under conditions suitable for expressing each chain of the antibody, and optionally recovering the antibody from the host cells (or the culture medium of the host cells).

[0360] 55. An immunoconjugate comprising a trispecific antibody as described in any one of Embodiments 1 to 50.

[0361] 56. A pharmaceutical composition, pharmaceutical, or preparation comprising a trispecific antibody according to any one of Embodiments 1 to 50 or an immunoconjugate according to Embodiment 55, and optionally a pharmaceutical auxiliary material.

[0362] 57. A pharmaceutical combination comprising a trispecific antibody described in any one of Embodiments 1 to 50 or an immunoconjugate described in Embodiment 55, and one or more additional therapeutic agents (e.g., chemotherapeutic agents).

[0363] 58. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a trispecific antibody described in any one of Embodiments 1 to 50, an immune conjugate described in Embodiment 55, a pharmaceutical composition or formulation described in Embodiment 56, or a pharmaceutical combination described in Embodiment 57.

[0364] 59. The method according to Embodiment 58, wherein the cancer is a GPRC5D-positive cancer, a BCMA-positive cancer, a GPRC5D / BCMA-double-positive cancer, or a cancer with dual low expression of BCMA and GPRC5D, for example, a cancer with low expression of GPRC5D and / or low expression of BCMA (e.g., a cancer that has recurred after anti-BCMA therapy or anti-GPRC5D therapy).

[0365] 60. A method according to Embodiment 58 or 59, wherein the cancer is a solid tumor or a hematological malignancy, for example, multiple myeloma.

[0366] 61. A method according to any one of embodiments 58 to 60, wherein the method further comprises the step of administering a therapeutic means (e.g., surgical therapy or radiotherapy) and / or an additional therapeutic agent (e.g., a chemotherapeutic agent) in combination.

[0367] In one aspect of the present invention, the present invention further relates to the following specific embodiments.

[0368] 1. A triplicate antibody that specifically binds to GPRC5D, comprising a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site that specifically binds to BCMA, wherein the first antigen-binding site comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein, The HCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO: 57; the HCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO: 58; the HCDR3 includes or consists of the amino acid sequence shown in SEQ ID NO: 59; the LCDR1 includes or consists of the amino acid sequence shown in SEQ ID NO: 61; the LCDR2 includes or consists of the amino acid sequence shown in SEQ ID NO: 62; A triplicate antibody in which the second antigen-binding region specifically binds to CD3 and / or the third antigen-binding region specifically binds to GPRC5D.

[0369] 2. A triplicate antibody according to Embodiment 1, wherein the VH of the first antigen-binding site that specifically binds to BCMA includes, or comprises, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in Sequence ID No. 56.

[0370] 3. A triplicate antibody according to Embodiment 1 or 2, wherein the VL of the first antigen-binding site that specifically binds to BCMA includes, or comprises, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 60.

[0371] 4. A triplicate antibody according to Embodiment 1, wherein the first antigen-binding site that specifically binds to BCMA comprises VH and VL, the VH consists of the amino acid sequence shown in SEQ ID NO: 56, and the VL of the first antigen-binding site consists of the amino acid sequence shown in SEQ ID NO: 60.

[0372] 5. A triplicate antibody according to any one of Embodiments 1 to 4, wherein the first antigen-binding site that specifically binds to BCMA comprises VH and VL, wherein VH comprises 39K and VL comprises 38D, or VH comprises 39D and VL comprises 38K.

[0373] 6. The triplicate antibody according to Embodiment 5, wherein the VH of the first antigen-binding site that specifically binds to BCMA is (i) containing the amino acid sequence shown in Sequence ID No. 76, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 76, and including an amino acid sequence containing the Q39K mutation, And the first antigen-binding site VL that specifically binds to BCMA is (i) containing the amino acid sequence shown in Sequence ID No. 75, or consisting of the said sequence, (ii) A triplicate antibody comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 75, and also comprising the Q38D mutation.

[0374] 7. A triplicate antibody according to any one of Embodiments 1 to 6, wherein the first antigen-binding site that specifically binds to BCMA is Fab.

[0375] 8. A trispecific antibody according to any one of Embodiments 1 to 7, wherein the third antigen-binding site that specifically binds to GPRC5D comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, where, The HCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 41; the HCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 42; the HCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 43; the LCDR1 contains or consists of the amino acid sequence shown in SEQ ID NO: 45; the LCDR2 contains or consists of the amino acid sequence shown in SEQ ID NO: 46; the LCDR3 contains or consists of the amino acid sequence shown in SEQ ID NO: 47; or The HCDR1 includes or consists of the amino acid sequence described in SEQ ID NO: 49; the HCDR2 includes or consists of the amino acid sequence described in SEQ ID NO: 50; the HCDR3 includes or consists of the amino acid sequence described in SEQ ID NO: 51; the LCDR1 includes or consists of the amino acid sequence described in SEQ ID NO: 53; the LCDR2 includes or consists of the amino acid sequence described in SEQ ID NO: 54; the LCDR3 includes or consists of the amino acid sequence described in SEQ ID NO: 55; Trispecific antibodies.

[0376] 9. The triplicate antibody according to Embodiment 8, wherein the VH of the third antigen-binding site that specifically binds to GPRC5D is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in Sequence ID No. 40, or an amino acid sequence consisting of such an amino acid sequence; or (ii) A trispecific antibody comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No. 48.

[0377] 10. A trispecific antibody according to Embodiment 8 or 9, wherein the VL of the third antigen-binding site that specifically binds to GPRC5D is (i) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in Sequence ID No. 44, or an amino acid sequence consisting of such an amino acid sequence; or (ii) A trispecific antibody comprising, or consisting of, an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in Sequence ID No. 52.

[0378] 11. A triplicate antibody according to Embodiment 8, wherein the third antigen-binding site that specifically binds to GPRC5D comprises VH and VL, wherein VH consists of the amino acid sequence shown in SEQ ID NO: 48 and VL consists of the amino acid sequence shown in SEQ ID NO: 52, or, where VH consists of the amino acid sequence shown in SEQ ID NO: 40 and VL consists of the amino acid sequence shown in SEQ ID NO: 44.

[0379] 12. A triplicate antibody according to any one of Embodiments 1 to 11, wherein the third antigen-binding site that specifically binds to GPRC5D comprises VH and VL, wherein VH comprises 39K and VL comprises 38D, or VH comprises 39D and VL comprises 38K.

[0380] 13. The triplicate antibody according to Embodiment 12, wherein the VH of the third antigen-binding site that specifically binds to GPRC5D is (i) containing the amino acid sequence shown in Sequence ID No. 80, or consisting of the said sequence, The amino acid sequence includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 80, and also includes the Q39K mutation. Furthermore, the VL of the third antigen-binding site includes or consists of the amino acid sequence shown in SEQ ID NO: 79, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 79, and also includes the Q38D mutation.

[0381] 14. A triplicate antibody according to any one of Embodiments 1 to 13, wherein the third antigen-binding site that specifically binds to GPRC5D is Fab.

[0382] 15. A triply specific antibody that specifically binds to GPRC5D, comprising a first antigen-binding site, a second antigen-binding site, and a third antigen-binding site that specifically binds to BCMA, wherein the first antigen-binding site comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, wherein, The HCDR1 contains or consists of the amino acid sequence described in SEQ ID NO: 65; the HCDR2 contains or consists of the amino acid sequence described in SEQ ID NO: 66; the HCDR3 contains or consists of the amino acid sequence described in SEQ ID NO: 67 or 73; the LCDR1 contains or consists of the amino acid sequence described in SEQ ID NO: 69; the LCDR2 contains or consists of the amino acid sequence described in SEQ ID NO: 70; the LCDR3 contains or consists of the amino acid sequence described in SEQ ID NO: 71; a triple-specific antibody.

[0383] 16. A triplicate antibody according to Embodiment 15, wherein the VH of the second antigen-binding site that specifically binds to CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 64 or 72, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the said sequence.

[0384] 17. A triplicate antibody according to Embodiment 15 or 16, wherein the VL of the second antigen-binding site that specifically binds to CD3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 60, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the said sequence.

[0385] 18. A triplicate antibody according to Embodiment 15, wherein the second antigen-binding site that specifically binds to CD3 comprises VH and VL, the VH consists of the amino acid sequence shown in SEQ ID NO: 64 or 72, and the VL of the second antigen-binding site consists of the amino acid sequence shown in SEQ ID NO: 68.

[0386] 19. A triplicate antibody according to any one of Embodiments 15 to 18, wherein the second antigen-binding site that specifically binds to CD3 comprises VH and VL, wherein VH comprises 39K and VL comprises 38D, or VH comprises 39D and VL comprises 38K.

[0387] 20. The triplicate antibody according to Embodiment 19, wherein the VH of the second antigen-binding site that specifically binds to CD3 is (i) containing the amino acid sequence shown in Sequence ID No. 77 or 78, or consisting of the said sequence, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 77 or 78, and containing the Q39D mutation; And the VL, the second antigen-binding site that specifically binds to CD3, (i) containing the amino acid sequence shown in Sequence ID No. 74, or consisting of the said sequence, (ii) A triplicate antibody containing an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 74, and containing the Q38K mutation.

[0388] 21. A triplicate antibody according to any one of Embodiments 1 to 20, wherein the second antigen-binding site that specifically binds to CD3 is Fab.

[0389] 22. A triplicate antibody according to any one of Embodiments 1 to 21, wherein the first antigen-binding site, the second antigen-binding site, and the third antigen-binding site are the first Fab, the second Fab, and the third Fab, respectively.

[0390] 23. A triplicate antibody according to Embodiment 22, wherein the first Fab or the third Fab further contains a disulfide bond remodeling mutation.

[0391] 24. A tripspecific antibody according to Embodiment 23, wherein the disulfide bond remodeling mutation comprises F126C in CH1 of Fab and Q124C in CL of Fab.

[0392] 25. The triplicate antibody according to Embodiment 24, wherein the Fab containing the disulfide bond remodeling mutation comprises CH1, and the CH1 is (i) Containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 85, and containing F126C, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 85.

[0393] 26. A triplicate antibody according to Embodiment 24 or 25, wherein the Fab containing a disulfide bond remodeling mutation includes CL, and the CL is (i) Containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 97, and containing Q124C, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 97.

[0394] 27. A triplicate antibody according to Embodiment 24, wherein Fab containing a disulfide bond remodeling mutation comprises CH1 and CL, wherein CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85, and CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 97.

[0395] 28. A tripspecific antibody according to Embodiment 24, wherein the disulfide bond remodeling mutations include F126C and C220S in CH1 of Fab, and Q124C and C214S in CL of Fab.

[0396] 29. The triplicate antibody according to Embodiment 28, wherein the Fab containing the disulfide bond remodeling mutation comprises CH1, and the CH1 is (i) Containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 86, and including F126C and C220S, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 86.

[0397] 30. A triplicate antibody according to Embodiment 28 or 29, wherein the Fab containing a disulfide bond remodeling mutation includes CL, and the CL is (i) Containing an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 98, and containing Q124C, or (ii) A triplicate antibody comprising or consisting of the amino acid sequence shown in Sequence ID No. 98.

[0398] 31. A triplicate antibody according to Embodiment 28, wherein Fab containing a disulfide bond remodeling mutation comprises CH1 and CL, wherein CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 86, and CL comprises or consists of the amino acid sequence shown in SEQ ID NO: 98.

[0399] 32. A triplicate antibody according to any one of Embodiments 1 to 31, wherein the triplicate antibody includes a first Fc region and a second Fc region, and the first Fc region and the second Fc region may be the same or different.

[0400] 33. A triplicate antibody according to Embodiment 32, wherein the first Fc region and the second Fc region are human IgG Fc, for example human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, and include, for example, the amino acid sequence shown in SEQ ID NO: 87 or 88, or an amino acid sequence having at least 90% (e.g., 95%, 96%, 97%, 99% or more) identity with said sequence, or consists of said amino acid sequence.

[0401] 34. A triplicate antibody according to Embodiment 33, wherein the first and / or second Fc region contains the L234A / L235A mutation.

[0402] 35. A triplicate antibody according to Embodiment 34, wherein the first and / or second Fc region comprises an amino acid sequence shown in SEQ ID NO: 89 or 90, or an amino acid sequence having at least 90% (e.g., 95%, 96%, 97%, 99% or more) identity with the sequence, and comprises the amino acid substitution L234A / L235A.

[0403] 36. A triplicate antibody according to any one of embodiments 32 to 35, wherein one of the first Fc region and the second Fc region contains a Knob mutation and the other contains a Hole mutation.

[0404] 37. A triplicate antibody according to Embodiment 36, wherein one Fc region contains the amino acid substitution T366W, and the other Fc region contains the amino acid substitutions T366S, L368A, and Y407V (according to EU number).

[0405] 38. A triplicate antibody according to Embodiment 37, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 92.

[0406] 39. A triplicate antibody according to Embodiment 32, wherein one Fc region comprises amino acid substitutions L234A / L235A and T366W, and the other Fc region comprises amino acid substitutions L234A / L235A, T366S, L368A and Y407V (according to EU number).

[0407] 40. A triplicate antibody according to Embodiment 39, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 94, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 93.

[0408] 41. A trispecific antibody according to any one of Embodiments 1 to 40, wherein the first antigen-binding site, the second antigen-binding site, and the third antigen-binding site are the first Fab, the second Fab, and the third Fab, respectively, (1) The first Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; and the third Fab fuses with the N-terminus of VH of the Fab heavy chain of the second Fab fragment at the C-terminus of CH1 of the Fab heavy chain; or The third Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; and the first Fab fuses with the N-terminus of VH of the Fab heavy chain of the second Fab fragment at the C-terminus of CH1 of the Fab heavy chain; or (2) The first Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, and the third Fab fuses with the C-terminus of the Fc region fused to the second Fab at the N-terminus of VH of the Fab heavy chain; or A third Fab fuses to one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; a second Fab fuses to another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain; and a first Fab fuses to the C-terminus of the Fc region fused to the second Fab at the N-terminus of VH of the Fab heavy chain; a triplicate antibody.

[0409] 42. A triplicate antibody according to Embodiment 41, wherein the fusion includes direct fusion or linker-mediated fusion.

[0410] 43. A triplicate antibody according to Embodiment 42, characterized in that the linker is (GGGGS)n and n=1, 2, 3, or 4.

[0411] 44. A triplicate antibody according to any one of Embodiments 41 to 43, wherein the second Fab comprises a charge mutation, the charge mutation comprising a Q38K or Q38D mutation in the Fab light chain and a Q39D or Q39K mutation in the Fab heavy chain.

[0412] 45. A triplicate antibody according to any one of Embodiments 41 to 44, wherein in the second Fab, or in the first or third Fab fused to the second Fab via the Fc region, the Fab heavy chain comprises the Q39K or Q39D mutation and the F126C mutation, and the Fab light chain comprises the Q38D or Q38K mutation and the Q124C mutation; or, in the second Fab, or in the first or third Fab fused to the second Fab via the Fc region, the Fab heavy chain comprises the Q39K or Q39D mutation and the F126C mutation and the C220S, and the Fab light chain comprises the Q38D or Q38K mutation and the Q124C mutation and the C214S.

[0413] 46. ​​A trispecific antibody according to any one of Embodiments 41 to 43, wherein, The second Fab contains a charge mutation, which includes a Q38K mutation in the Fab light chain and a Q39D mutation in the Fab heavy chain, and in the first or third Fab fused to the second Fab, or to the second Fab via the Fc region, the Fab heavy chain contains Q39K and F126C mutations, and the Fab light chain contains Q38D and Q124C mutations; or, The second Fab contains a charge mutation, which includes a Q38K mutation in the Fab light chain and a Q39D mutation in the Fab heavy chain, and in the first or third Fab fused to the second Fab, or to the second Fab via the Fc region, the Fab heavy chain contains Q39K and F126C mutations and C220S, and the Fab light chain contains Q38D and Q124C mutations and C214S; or, The second Fab contains a charge mutation, which includes a Q38D mutation in the Fab light chain and a Q39K mutation in the Fab heavy chain, and in the first or third Fab fused to the second Fab, or to the second Fab via the Fc region, the Fab heavy chain contains Q39D and F126C mutations, and the Fab light chain contains Q38K and Q124C mutations; or, A triplicate antibody comprising a second Fab containing charge mutations, which include a Q38D mutation in the Fab light chain and a Q39K mutation in the Fab heavy chain, and a first or third Fab fused to the second Fab, or to the second Fab via the Fc region, wherein the Fab heavy chain contains Q39D and F126C mutations and C220S, and the Fab light chain contains Q38K and Q124C mutations and C214S.

[0414] 47. A triplicate antibody according to any one of Embodiments 1 to 46, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer consisting of five polypeptide chains, and the following peptide chains This is a peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to GPRC5D, Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, A peptide chain 3# having a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain in sequence, Peptide chain 4# containing a Fab light chain that specifically binds to BCMA, and Peptide chain 5# containing a Fab light chain that specifically binds to CD3 Composed of, In this triple-specific antibody, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form a third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the first Fab and second Fab, respectively.

[0415] 48. A trispecific antibody as described in Embodiment 47, wherein, Peptide chain 1# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4; Peptide chain 3# contains, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 3 or 6; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2; and / or A triplicate antibody comprising peptide chain 5# containing, or consisting of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0416] 49. A trispecific antibody according to Embodiment 47, wherein, Peptide chain 1# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 15, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 15; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 14, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 14; Peptide chain 3# contains, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence shown in SEQ ID NO: 3 or 6; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2; and / or A triplicate antibody comprising peptide chain 5# containing, or consisting of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0417] 50. A triplicate antibody according to any one of Embodiments 1 to 46, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer, consisting of five polypeptide chains, the following peptide chains: Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to BCMA, Peptide chain 2# containing a Fab heavy chain that specifically binds to BCMA, A Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and a peptide chain 3# having Fc domains in sequence, Peptide chain 4# containing a Fab light chain that specifically binds to GPRC5D, and Peptide chain 5# containing a Fab light chain that specifically binds to CD3 It consists of, In this triple-specific antibody, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the first Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the third Fab and second Fab, respectively.

[0418] 51. A triplicate antibody according to Embodiment 50, wherein, Peptide chain 1# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 10, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10; Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 9, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 9; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 8; and / or A triplicate antibody comprising peptide chain 5# containing, or consisting of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1.

[0419] 52. A trispecific antibody according to any one of Embodiments 1 to 46, wherein the trispecific antibody is a left-right asymmetric IgG-like pentamer, consisting of five polypeptide chains, the following peptide chains: Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to GPRC5D, Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain 3# sequentially comprises a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain of an antigen-binding region that specifically binds to BCMA. Peptide chain 4# containing a Fab light chain that specifically binds to CD3, and Peptide chain 5# containing the Fab light chain, which is an antigen-binding site that specifically binds to BCMA. It consists of, In this triple-specific antibody, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the second Fab and first Fab, respectively.

[0420] 53. A triplicate antibody according to Embodiment 52, wherein, Peptide chain 1# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 5, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5; Peptide chain 2# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4; Peptide chain 3# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7; Peptide chain 4# contains, or consists of, the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1; and / or A triplicate antibody comprising, or consisting of, a peptide chain 5# containing, the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2.

[0421] 54. A nucleic acid molecule encoding any of the chains of the trispecific antibody described in any one of Embodiments 1 to 53, or a nucleic acid molecule consisting of a nucleic acid sequence.

[0422] 55. An expression vector comprising a nucleic acid molecule as described in Embodiment 54, wherein the expression vector is, for example, a pCNDA vector (e.g., a pcDNA3.1 expression vector).

[0423] 56. A host cell comprising the nucleic acid molecule described in Embodiment 54 or the expression vector described in Embodiment 52, wherein the host cell is preferably a prokaryotic or eukaryotic cell, for example, a 293 cell or a CHO cell (e.g., a HEK293 cell).

[0424] 57. A method for producing a tripspecific antibody according to any one of Embodiments 1 to 53, the method comprising culturing host cells containing a nucleic acid molecule according to Embodiment 54 or an expression vector according to Embodiment 55 under conditions suitable for expressing each chain of the antibody, and optionally recovering the antibody from the host cells (or the culture medium of the host cells).

[0425] 58. An immunoconjugate comprising a trispecific antibody as described in any one of Embodiments 1 to 53.

[0426] 59. A pharmaceutical composition, pharmaceutical, or preparation comprising a trispecific antibody according to any one of Embodiments 1 to 53 or an immunoconjugate according to Embodiment 58, and optionally a pharmaceutical auxiliary material.

[0427] 60. A pharmaceutical combination comprising a trispecific antibody described in any one of Embodiments 1 to 53 or an immunoconjugate described in Embodiment 55, and one or more additional therapeutic agents (e.g., chemotherapeutic agents).

[0428] 61. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a trispecific antibody described in any one of Embodiments 1 to 53, an immune conjugate described in Embodiment 58, a pharmaceutical composition or formulation described in Embodiment 59, or a pharmaceutical combination described in Embodiment 57.

[0429] 62. The method according to Embodiment 61, wherein the cancer is a GPRC5D-only positive cancer, a BCMA-only positive cancer, a GPRC5D / BCMA double-positive cancer, or a cancer with dual low expression of BCMA and GPRC5D, for example, a cancer with low expression of GPRC5D and / or low expression of BCMA (e.g., a cancer that has recurred after anti-BCMA therapy or anti-GPRC5D therapy).

[0430] 63. A method according to Embodiment 61 or 62, wherein the cancer is a solid tumor or a hematological malignancy, for example, multiple myeloma.

[0431] 64. A method according to any one of embodiments 58 to 60, the method further comprising the step of administering a therapeutic means (e.g., surgical therapy or radiotherapy) and / or an additional therapeutic agent (e.g., a chemotherapeutic agent) in combination.

[0432] It should be understood that the present invention also encompasses any combination of any technical features or any combination of any technical solutions described herein.

[0433] Examples Example 1. Molecular structure design and construction of a triple-specific antibody (Ts) The antibody in this embodiment is a multispecific antibody of the T-cell engager class that simultaneously targets GPRC5D, BCMA, and CD3, and can simultaneously bind to two tumor-associated antigens on the surface of multiple myeloma (MM) cells (i.e., GPRC5D and BCMA) and the CD3 receptor on the surface of T cells. The multispecific antibody that simultaneously targets GPRC5D, BCMA, and CD3, and includes disulfide-binding remodeling mutations and charge mutations, was designed based on two anti-GPRC5D antibodies with different affinities (hz7F5.3 and hz5E12.1.P1), one anti-BCMA antibody (ADI-38497), and two anti-CD3 antibodies with different affinities (hzsp34.24 and hzsp34.87). The sequence numbers and specific sequences of the variable regions and CDR sequences of the above three antibodies are shown in the sequence information.

[0434] Specifically, in this invention, six example antibodies (B3, B3b, B5) with the 1+1+1 structure shown in Figure 1 and Table 1 were designed, achieving excellent activity against bispecific antibodies. Furthermore, in the design of the example antibodies, light chain mispairing in Fab other than BCMA and CD3Fab was resolved using specific mutation methods (disulfide bond remodeling mutations and charge mutations; see sequence information table for specific mutation information). In addition, heavy chain mispairing in the left-right asymmetric IgG-like bispecific antibody at the Fc site was resolved using the Knob-in-Hole method. In this case, Fc is the heavy chain constant region of IgG1 into which amino acid mutations L234A and L235A (EU numbering) that reduce effector function have been introduced.

[0435] The design structures of the six example antibodies described above are shown in Figure 1 and Table 1.

[0436] Table 1. Fragment numbers and descriptions of example antibodies

[0437] [Table 1]

[0438] The structures of the six example antibodies described above are in B3 format, B3b format, and B5 format.

[0439] The B3 format (see Figure 1A for details such as chain number) is an asymmetrical IgG-like pentamer consisting of five polypeptide chains, with the following peptide chains: Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to GPRC5D, Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, A peptide chain 3# having a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain in sequence, Peptide chain 4# containing a Fab light chain that specifically binds to BCMA, and Peptide chain 5# containing a Fab light chain that specifically binds to CD3 It consists of, Here, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the first Fab and second Fab (as shown in Figure 1A [B3 format] for example).

[0440] The B3b format (see Figure 1A for details such as chain number) is an asymmetrical IgG-like pentamer consisting of five polypeptide chains, with the following peptide chains: Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to BCMA, Peptide chain 2# containing a Fab heavy chain that specifically binds to BCMA, A Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and a peptide chain 3# having Fc domains in sequence, Peptide chain 4# containing a Fab light chain that specifically binds to GPRC5D, and Peptide chain 5# containing a Fab light chain that specifically binds to CD3 It consists of, Here, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the first Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the third Fab and second Fab (as shown in Figure 1A [B3b format] as an example).

[0441] The B5 format (see Figure 1B for details such as chain number) is an asymmetrical IgG-like pentamer consisting of five polypeptide chains, and the following peptide chains: Peptide chain 1# having a Fab heavy chain and an Fc domain that specifically bind to GPRC5D, Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain 3# having sequentially a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain with an antigen-binding site that specifically binds to BCMA. Peptide chain 4# containing a Fab light chain that specifically binds to CD3, and Peptide chain 5# containing the Fab light chain, which is an antigen-binding site that specifically binds to BCMA. It consists of, Here, the Fab heavy chain of peptide chain 1# pairs with the Fab light chain of peptide chain 2# to form the third Fab, and the two Fab heavy chains of peptide chain 3# pair with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the second Fab and the first Fab (as shown in Figure 1B for example).

[0442] Furthermore, in this patent, the following bispecific or trispecific antibodies were expressed and purified: Inno-B (control Hel / BCMA / CD3, where Hel is a chicken egg white lysozyme antibody that does not bind to any human-related protein and was used as a control), Inno-G (control Hel / GPRC5D / CD3), F2(24) (GPRC5D / BCMA / CD3, structure derived from WO2022174813A1, BCMA antigen binding site is scFv), F2(87) (GPRC5D / BCMA / CD3, structure derived from WO2022174813A1, BCMA antigen binding site is scFv), JNJ-BCMA / CD3 (BCMA / CD3, derived from patent WO2017031104A1, also known as Teclistamab), Roch e-BCMA / CD3 (BCMA / CD3, derived from patent WO2018083204A1, also known as Alnuctamab), REGN-BCMA / CD3 (BCMA / CD3, derived from patent US20200024356A), JNJ-GPRC5D / CD3 (GPRC5D / CD3, derived from patent WO2018017786A2), Roche-GPRC5D / CD3 (GPRC5D / CD3, derived from patent WO2019 / 154890 A1), Elranatamab (Pfizer's BCMA×CD3, derived from patent WO2016166629A1).

[0443] Furthermore, the following negative control antibody was also expressed and purified. This was an anti-Hel antibody (indicated as IgG or hIgG1 in the figure), with its heavy chain sequence being SEQ ID NO: 101 and its light chain sequence being SEQ ID NO: 100.

[0444] Example 2. Preparation and purification of triplicate antibodies Plasmid preparation The nucleic acids encoding each chain of each antibody of the present invention were synthesized using GENEWIZ and individually constructed on pCDNA3.1 template plasmids. A predetermined amount of plasmid was prepared, filtered through a 0.22 μm filter head, and subjected to transient expression in cells.

[0445] Transfection and Expression Expi293 cells (Invitrogen) were passaged according to the desired transfection volume. The cell concentration was increased to 3 × 10⁻⁶ the day before transfection. 6 The cell concentration was adjusted to cells / mL. On the day of transfection, the cell concentration was also adjusted to 3 × 10⁻¹⁶. 6 The solution was readjusted to cells / mL. At the time of expression, each molecule was dispensed into two flasks, A and B. Plasmids containing peptide chains 1# and 2# were added to flask A in a 1:1 ratio, and plasmids containing peptide chains 3#, 4#, and 5# were added to flask B in a 1:1:1 ratio. Opti-MEM medium (Gibco, Cat. No. 31985-070) was used as the transfection buffer, and the plasmids were diluted to 1 / 10 (v / v) of the final volume and thoroughly mixed. An appropriate amount of polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmids from the previous step (mass ratio of plasmid to PEI was 1:3), thoroughly mixed, and incubated at room temperature for 10 minutes to obtain a DNA / PEI mixture. The DNA / PEI mixture was gently added to HEK293 cells, thoroughly mixed, and incubated at 37°C with 8% CO2 for 24 hours. Then, VPA (Sigma, Cat.No.P4543-100G) was added to a final concentration of 2 mM. Subsequently, a 2% (v / v) feed (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone) was added, and the cells were incubated for another 6 days.

[0446] purification The culture medium was collected and centrifuged at 4000 rpm for 30 minutes. The supernatant was collected, filtered through a 0.45 μm filter membrane, and purified by Protein A affinity chromatography, then by KappaSelect affinity chromatography, and finally by LambdaFabSelect affinity chromatography. Finally, in vitro recombination was performed, and the complete molecule was purified by ion exchange chromatography.

[0447] Protein A affinity chromatography was performed as follows. The supernatant was purified using a pre-packed HiTrap MabSelect Sure (GE, 11-0034-95) column. The procedure was as follows: Before purification, the column was equilibrated with 5 column volumes of equilibration buffer (20 mM Tris, 150 mM NaCl, pH 7.2). After passing the collected supernatant through the column, it was washed with 10 column volumes of equilibration buffer to remove nonspecific binding proteins. Subsequently, the column was eluted with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and the eluted fraction was collected. 2 M Tris was added for neutralization to pH 6.5. The correct pairing ratio of the purified antibody was determined by liquid chromatography-mass spectrometry (LC-MS).

[0448] KappaSelect affinity chromatography was performed as follows. The supernatant was purified using a pre-packed HiTrap KappaSelect column (GE, 17-5458-11). The procedure was as follows: Before purification, the column was equilibrated with 5 column volumes of equilibration buffer (PBS, pH 7.4). After passing the collected supernatant through the column, it was washed with 10 column volumes of equilibration buffer to remove nonspecific binding proteins. Subsequently, the column was eluted with 5 column volumes of elution buffer (0.1 M glycine buffer, pH 2.5), and the eluted fraction was collected. 2 M Tris was added for neutralization to bring the pH to 6.5. The correct pairing ratio of the purified antibody was determined by liquid chromatography-mass spectrometry (LC-MS).

[0449] LambdaFabSelect affinity chromatography was performed as follows. The supernatant was purified using a pre-packed HiTrap LambdaFabSelect column (GE, 17-5482-11). The procedure was as follows: Before purification, the column was equilibrated with 5 column volumes of equilibration buffer (PBS, pH 7.4), and the recovered supernatant was passed through it. Then, it was washed with 10 column volumes of equilibration buffer to remove nonspecific binding proteins. Subsequently, elution was performed with 5 column volumes of elution buffer (0.1M acetate buffer, pH 3.5), and the eluted fraction was collected. 2M Tris was added for neutralization to pH 6.5. The correct pairing ratio of the purified antibody was determined by liquid chromatography-mass spectrometry (LC-MS).

[0450] in vitro recombination In vitro recombination was performed as follows: Molecule A obtained in flask A and molecule B obtained in flask B were mixed in a molar ratio of 1:1. GSH (Sigma Aldrich, G4251) at a final concentration of 5 mM and arginine (Sigma Aldrich, A5006) at a final concentration of 50 mM were added, and the mixture was incubated at 37°C for 6 hours.

[0451] Refining (final refining) Purification by ion exchange chromatography was performed as follows: The product obtained by in vitro recombination was buffer-exchanged to pH 6.0 low-salt PB buffer (10 mM phosphate, pH 6.0) using an ultrafiltration concentration tube (MILLIPORE, Cat. No. UFC901096), and then subjected to final purification using Capto HiRes S 10 / 100 (GE, Cat. No. 29275879). Before purification, the packed column was equilibrated with 5 column volumes of pH 6.0 low-salt PB buffer, and the buffer-exchanged sample was passed through the column. Subsequently, to remove nonspecific binding proteins, the column was washed with 10 column volumes of the same buffer. Elution was performed by linearly increasing the concentration of pH 6.0 high-salt PB buffer (10 mM phosphate, pH 6.0, 1 M NaCl) (linear gradient: increasing the ratio of pH 6.0 high-salt PB buffer from 1% to 100% over 30 column volumes), and the main elution peak was recovered. The buffer solution was replaced with PBS (Gibco, Cat. No. 70011-044) using an ultrafiltration concentration tube (MILLIPORE, Cat. No. UFC901096).

[0452] Figure 7 shows the differences in aggregates between the B3(24) molecule and the control molecule F2(24).

[0453] The B3(24) molecule of the present invention exhibited significantly improved protein properties compared to the triple-specific antibody F2(24) described in patent application WO2022174813A1. This is supported by the fact that the proportion of aggregates generated during cation exchange chromatography purification after in vitro recombination was greatly reduced. As evaluated by cation exchange chromatography, the triple-specific antibody F2(24) of patent application WO2022174813A1 showed approximately 35% aggregates after in vitro recombination (Figure 7A), whereas the B3(24) molecule showed very few or virtually no aggregates after in vitro recombination (Figure 7B). Since aggregates are a type of impurity that needs to be removed in the purification process, a significant reduction in aggregates leads to a remarkable improvement in the recovery rate in the purification process.

[0454] Example 3. Measurement of affinity of a trispecific antibody against antigen-expressing cells. 3.1 The affinity of the triplicate antibody against antigen-expressing cells was measured.

[0455] The trispecific antibodies in the examples were assayed using the GS-CHO-hBCMA and GS-CHO-cynoBCMA cell lines for affinity to human (h-) and cynomolgus monkey (cyno-) BCMA, the GS-CHO-hGPRC5D and GS-CHO-cynoGPRC5D cell lines for affinity to human (h-) and cynomolgus monkey (cyno-) GPRC5D, and the GS-CHO-hGPRC5D and GS-CHO-cynoGPRC5D cell lines for affinity to human CD3.

[0456] GS-CHO cell lines overexpressing GPRC5D (GS-CHO-hGPRC5D and GS-CHO-cynoGPRC5D) were constructed as follows: Nucleic acids encoding the full-length sequences of human and monkey GPRC5D (human GPRC5D: Q9NZD1, UniProt; monkey GPRC5D: A0A2K5W6I7, UniProt) were inserted into PEE17.4 plasmids (Lonza, GS Xceed Expression System), and the constructed stable expression cell lines were screened under selective pressure.

[0457] GS-CHO cell lines overexpressing BCMA (GS-CHO-hBCMA and GS-CHO-cynoBCMA) were constructed as follows: Nucleic acids encoding the full-length sequences of human and monkey BCMA (human BCMA: Q02223, UniProt; monkey BCMA: A0A2K5UD97, UniProt) were inserted into T2A-EGFP plasmids (GENEWIZ), and the constructed stable-expressing cell lines were screened under selective pressure.

[0458] Specifically, cultured cells were obtained according to the instructions for use, or T cells were isolated from human PBMCs and seeded at 1E5 cells / well in a 96-well V-bottom plate. Next, the test molecule and control molecule were serially diluted (see Figure 2 for details) and co-incubated with cells expressing the corresponding antigen at 4°C for 40 minutes. Subsequently, a fluorescently labeled secondary antibody (1:200 dilution; also mixed with DCM at 1:2000 as a cell viability indicator) was added. Isolated human T cells were labeled with CD4 and CD8 antibodies (1:200 dilution each) to identify the relevant T cells. Finally, the fluorescence intensity of the cells was detected using a flow cytometer (BD), and the EC50 value was calculated by curve fitting using GraphPad Prism 8.0. Figures 2A-H and Tables 2-9 show the binding affinity of the antibodies of the present invention to the corresponding antigens. Reagents and Materials

[0459] [Table 2]

[0460] The specific results are shown in Figure 2 and Tables 2-10.

[0461] Table 2 Affinity of the example antibody against human BCMA - 1

[0462] [Table 3]

[0463] ND: Not detected Table 3 Affinity of the example antibody against human BCMA - 2

[0464] [Table 4]

[0465] Table 4 Affinity of the antibody used in the example against cynomolgus monkey BCMA - 1

[0466] [Table 5]

[0467] Table 5 Affinity of the example antibody against human GPRC5D - 1

[0468] [Table 6]

[0469] Table 6 Affinity of the example antibody against human GPRC5D - 2

[0470] [Table 7]

[0471] Table 7 Affinity-1 of the example antibody against cynomolgus monkey GPRC5D

[0472] [Table 8]

[0473] Table 8 Affinity of the example antibody against human CD3 - 1

[0474] [Table 9]

[0475] Table 9 Affinity of the example antibody against human T cells

[0476] [Table 10]

[0477] 3.2 Measurement of affinity of triplicate antibodies against mutant antigen-expressing cells The methods for obtaining 293T cell lines (293T-hBCMA-P34del, 293T-hBCMA-S30del, 293T-hBCMA-R39A, 293T-hBCMA-R27P) overexpressing BCMA variants and GS-CHO cell lines (GS-CHO-hBCMA-P34del, GS-CHO-hBCMA-S30del, GS-CHO-hBCMA-R39A, GS-CHO-hBCMA-R27P) overexpressing BCMA variants are as follows. Corresponding point mutations or deletion mutations were introduced into the full-length sequence of human BCMA (human BCMA (Q02223, Uniprot database)). The obtained mutant coding nucleic acids were respectively inserted into the T2A-EGFP plasmid (GENEWIZ), and pressure screening (selection pressure screening) was performed on the constructed stable expression cell lines. The amino acid sequences and coding nucleic acid sequences of the human BCMA full-length sequence, hBCMA-P34del, hBCMA-S30del, hBCMA-R39A and hBCMA-R27P are described in the sequence information.

[0478] 1. Specific construction method: After the 293T cells were revived, they were passaged 2 - 3 times. After removing the medium, they were washed with 10 mL of PBS and digested with 2 mL of trypsin. After stopping the digestion with the medium, they were centrifuged at 400×g for 3 minutes and the supernatant was removed. The cells were resuspended in the medium, and 20 μL of the cell suspension was aliquoted into a 1.5 mL EP tube. The cell count was measured, and 7 seeded with 1.5×10 cells in a T175 flask. The cells were gently shaken to homogenize and placed horizontally in an incubator at 37 °C for overnight culture. The pre-seeded 293T cells were taken out, and after removing the medium, 20 mL of DMEM medium containing 10% FBS and 1% PS was added to each flask, thereby obtaining transfection mixture 1.

[0479]

Table 11

[0480] Mixture 1 was slowly added dropwise to mixture 2 all at once, and after thorough mixing, it was allowed to stand at room temperature for 15 minutes.

[0481] The transfection mixture was added to the culture medium for 293T cells, the flask was labeled, and the mixture was shaken to ensure uniform distribution of the transfection mixture throughout the medium. After 4–6 hours post-transfection, the medium was discarded and 20 mL of fresh DMEM supplemented with 2% FBS was added. At 48 and 72 hours post-transfection, the cell culture supernatant was collected, centrifuged at 4°C and 500×g for 10 minutes, and filtered through a 0.45 μm low-protein-binding filter membrane. After removing the supernatant, the cells were digested with 2 mL of trypsin, digestion was stopped with culture medium, the cells were centrifuged at 400 × g for 3 minutes, the supernatant was discarded, the cells were resuspended in culture medium and transferred to culture flasks to obtain BCMA mutant overexpressing 293T cell lines (293T-hBCMA-P34del, 293T-hBCMA-S30del, 293T-hBCMA-R39A, 293T-hBCMA-R27P), which were then continuously cultured in an incubator. The viral supernatant was mixed with Takara's virus concentrate in a ratio of 3:1 (v / v), sealed with Parafilm, and incubated overnight at 4°C; after incubation, the mixture was centrifuged at 4°C and 1500×g for 45 minutes, and the supernatant was discarded; the resulting lentivirus pellet was resuspended in 1 mL of CD CHO medium, held at 4°C until completely soluble, then aliquoted and stored at -80°C.

[0482] Construction of BCMA mutant overexpressing GS CHO cell line: GS CHO cells were centrifuged at 400 × g for 4 minutes, the supernatant was discarded, and the cells were resuspended in 3 mL of complete medium. The number of cells was counted, and 1 × 10⁶ cells were placed in a 6-well plate. 6Cells were seeded at a rate of one cell per well, and the virus supernatant was brought into contact with the resuspended cells in a ratio of 2:1. The culture medium was replenished to 1 mL per well, and after 4 hours of infection, an additional 2 mL was added to each well. After 24 hours of infection, the cells were collected in a centrifuge tube and centrifuged at 400 × g for 4 minutes. The virus-containing medium was removed and the cells were resuspended in fresh CD CHO medium containing 10% FBS and 1% PS. The cells were seeded in a T25 flask and cultured upright in a CO2 incubator. After culturing the cells for 48 hours, the culture medium was replenished to 10 mL and the cells were continued to be cultured in a CO2 incubator. CHO cells cultured at 37°C were removed and collected by centrifugation at 400 × g for 4 minutes. The supernatant was discarded, and the cells were inoculated into 125 mL Erlenmeyer flasks and cultured in a 37°C constant temperature shaking incubator to obtain BCMA mutant overexpressing GS CHO cell lines (GS CHO-hBCMA-P34del, GS CHO-hBCMA-S30del, GS CHO-hBCMA-R39A, GS CHO-hBCMA-R27P). 2. Detection method: Cultured BCMA mutant overexpressing 293T cell lines (293T-hBCMA-P34del, 293T-hBCMA-S30del, 293T-hBCMA-R39A, 293T-hBCMA-R27P) were removed, the culture medium was discarded, the cells were washed with 10 mL of PBS, and then digested with 2 mL of trypsin; digestion was stopped with culture medium, the cells were centrifuged at 400 × g for 5 minutes, and the supernatant was discarded.

[0483] Cells were resuspended in culture medium, and 20 μL of the cell suspension was dispensed into a 1.5 mL EP tube. Cell numbers were counted, and cells were seeded at a rate of 1E5 cells / well in a 96-well U-bottom plate. The test antibody and control molecule were serially diluted. The antibody was diluted 3-fold in PBS from an initial concentration of 1500 nM, resulting in a total of 12 dilutions. 50 μL of the serially diluted sample was incubated with seeded cells at 4°C for 30 minutes, and then a fluorescently labeled secondary antibody (Anti-human Fc-PE, Abcam, ab98596, 1:200 dilution) was added and incubated at 4°C for 30 minutes. Finally, cell fluorescence intensity was measured by flow cytometry (BD), and curve fitting was performed using GraphPad Prism 8.0. 50 The value was calculated.

[0484] GS CHO cell lines overexpressing BCMA mutants (GS CHO-hBCMA-P34del, GS CHO-hBCMA-S30del, GS CHO-hBCMA-R39A, GS CHO-hBCMA-R27P) were removed from culture, centrifuged at 400×g for 5 minutes, the supernatant was discarded, and the cells were resuspended in culture medium. 20 μL of the cell suspension was transferred to a 1.5 mL EP tube. The cells were counted and seeded at a rate of 1E5 cells / well in a 96-well U-bottom plate. The test molecule and control molecule were then serially diluted. Antibodies were diluted 3-fold in PBS from an initial concentration of 800 nM, for a total of 12 dilutions. 50 μL of the serially diluted drug was incubated with seeded cells at 4°C for 30 minutes, and then fluorescently labeled secondary antibody (1:200 dilution) was added and incubated at 4°C for 30 minutes. Finally, cell fluorescence intensity was measured by flow cytometry (BD), and curve fitting was performed using GraphPad Prism 8.0. 50 The value was calculated.

[0485] 3.3 Experimental Results: Figures 2I and 2J show the following results: B3(24) can bind to wild-type BCMA-overexpressing cells, BCMA-P34del-overexpressing cells, BCMA-P30del-overexpressing cells, BCMA-R39A-overexpressing cells, and BCMA-R27P-overexpressing cells. Alnuctamab (Roche-BCMA×CD3) can bind to wild-type BCMA-overexpressing cells, BCMA-P34del-overexpressing cells, BCMA-P30del-overexpressing cells, BCMA-R39A-overexpressing cells, and BCMA-R27P-overexpressing cells. Elranatamab (Pfizer BCMA×CD3) can bind to wild-type BCMA-overexpressing cells and BCMA-P34del-overexpressing cells, and weakly binds to BCMA-R39A-overexpressing cells.

[0486] Teclistamab / JNJ-BCMA / CD3 (J&J BCMA×CD3) can weakly bind to wild-type BCMA-overexpressing cells and very weakly bind to BCMA-R39A-overexpressing cells.

[0487] Example 4. Affinity measurement of triplicate antibodies using BLI. The affinity of the anti-BCMA end and anti-CD3 end of the triply specific example antibody of the present invention was detected by biolayer interferometry (BLI). In this invention, the binding dynamics of the example antibody against human BCMA, cynomolgus monkey BCMA, human CD3 E&G, and cynomolgus monkey CD3 E&G were measured based on BLI using an optical fiber biosensor. When biomolecules bind to the sensor surface, a biofilm layer is formed, causing interference in the waveform of light transmitted through the sensor. This interference is detected as a phase shift, allowing us to understand the change in the number of molecules bound to the sensor. A dynamic curve was fitted from the changes in the obtained real-time response values, k on , k off and K D The result was calculated.

[0488] Specifically, sensors functionalized with streptavidin (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). The example antibody and biotin-labeled or Fc-tagged BCMA and CD3 E&G heterodimer antigens were diluted in SD buffer, respectively. 200 μL of SD buffer was dispensed, and each diluted sample (100 nM) was individually aliquoted into a 96-well black plate. The sensors and samples were placed in an Octet (Fortebio, Red96e). Data Acquisition 10.0 was launched, and "New Kinetic Experiment" was selected. The sensors were positioned corresponding to the sample locations, and the operation procedure and time were set to Baseline 60 s, Loading 250 s, Baseline 100 s, Association 600 s, and Dissociation 600 s. The experiment was conducted at 1000 rpm and 30°C.

[0489] The results were analyzed using the "Data Analysis 10.0" software, and the reference channel of the buffer was subtracted. A 1:1 binding model was selected for data fitting, and the kon, koff, and KD of the example antibody were calculated.

[0490] Antigen information

[0491] [Table 12]

[0492] Reagent and material information

[0493] [Table 13]

[0494] Table 10 shows the experimental results obtained by following the assay procedure described above. The results indicate that the affinity of the four example antibodies in B3 format for BCMA and CD3 is similar to that of the F2 antibody.

[0495] Table 10 Affinity of example antibodies detected by BLI

[0496] [Table 14]

[0497] ND: Not detected Example 5. Killing assay of human multiple myeloma cells and cytokine release using a triplicate antibody. When polyclonal antibodies bind to GPRC5D and / or BCMA on the surface of MM cells and also to CD3E on the surface of primary T cells, cross-linking occurs with T cells in a tumor-associated antigen (TAA, i.e., GPRC5D and / or BCMA)-dependent manner, inducing T cell activation and thereby mediating the killing of tumor cells. In actual multiple myeloma patients, due to the high heterogeneity of tumor cells expressing BCMA and GPRC5D, specificity manifests as the existence of different subtypes of tumor cells expressing one or more antigens, or as individual differences in the antigen levels expressed by tumor cells.

[0498] Generation of KO cell lines and flow cytometry H929 ko BCMA and H929 ko GPRC5D were generated by transfection of H929 cells using the RNP method. gRNAs with high MIT specificity scores (Genescript) were selected using the gRNA design website (broadinstitute.org). Chemosynthetic gRNAs (three per target protein) were mixed with Cas9 protein (Genscript SC1841 NA) and introduced into cells by electrotransfection. After stable culture was achieved, primer PCR was designed near the gRNA cleavage sites, and the amplified products were sequenced. After Sanger sequencing (Genescript), cleavage efficiency was evaluated by CRISPR analysis, and the cleavage ratio of the target gene was calculated. The knockout cell pool was labeled with BCMA and GPRC5D using a flow cytometry cell sorter (BD), and the cell population negative for the target protein was sorted. Results: The final selected gRNA sequences and flow cytometry results of the obtained cell lines are shown in the table and Figure 6 below, respectively.

[0499] gRNA sequences selected in H929 knockout cell lines

[0500] [Table 15]

[0501] Killing assay and cytokine release To further investigate the killing effect of this antibody in multiple myeloma, 1) cells expressing a single TAA, namely H929 ko BCMA and H929 ko GPRC5D (prepared as described above), and 2) H929 cells (MM cells, Nanjing Cobioer, CBP60243) highly expressing a dual TAA or L363 cells (MM cells, Nanjing Cobioer, CBP60240) low in expression of a dual TAA were co-cultured with PBMCs (Saily, SLB-HP050B) at an effector cell to tumor cell ratio of 10:1. The antibody in this example and the control antibody were added, and dead cells were detected after 16 hours by lactate dehydrogenase (LDH) release assay. Non-target cells Calu-6 (NSCLC cells, Chinese Academy of Sciences Cell Library, TCHu144) that do not express both antigens were co-cultured with the corresponding antibody and PBMCs and used as a control for nonspecific killing. 3) H929 KO GPRC5D and H929 KO BCMA expressing a single TAA were mixed with parental H929 cells expressing both antigens in a 1:1:1 ratio and then co-cultured with PBMCs. The antibody used in this example and the control antibody were added, and the viability of target tumor cells was analyzed by flow cytometry after 16 hours.

[0502] Reagents and materials:

[0503] [Table 16]

[0504] The experimental procedure was as follows: PBMC cells (Saily, SLB-HP050B) were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and added to 9 mL of serum-free medium. The mixture was centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cells were resuspended in phenol red-free 1640 medium with 10% FBS, and the cell density was increased to 2 × 10⁶. 6The cell density was adjusted to cells / mL. Tumor cells in the logarithmic growth phase were collected and washed twice with PBS to remove excess serum. At this point, if flow cytometry was to be used for detecting cell death, CTV staining solution (1 μM, CellTrace (registered trademark Violet DMSO stock solution: PBS = 1:50,000; 2 × 10^6 cells / mL CTV staining solution) was added after collecting the MM cells, and the mixture was incubated at 37 °C in the dark for 15 minutes. On the other hand, if LDH was to be used for detecting tumor cell death, this step was omitted, and centrifugation was performed immediately. The mixture was centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and the cell pellet was retained. The cells were resuspended in phenol red-free medium, and the cell density was adjusted to 4 × 10^6 cells. 5 The solution was adjusted to cells / mL. Depending on the requirements of the cell type, the antibody was serially diluted 10-fold in complete medium to obtain a total of eight concentrations (initial concentration of 200 nM and maximum final concentration of 50 nM for H929 cells (including KO cells), initial concentration of 400 nM and maximum final concentration of 100 nM for L363 and Calu-6), in addition to setting a final concentration of 0. 50 μL of tumor cells, 50 μL of serially diluted antibody, and 100 μL of PBMC cells were added to a 96-well cell culture plate (U-bottom, COSTAR, Cat No. 7007) and co-cultured. When detecting tumor cell death by LDH, a target cell spontaneous LDH-releasing well (50 μL of tumor cells only) and a target cell maximum LDH-releasing well (50 μL of tumor cells with lysis solution (LDH kit included, 1:10 dilution) added) were prepared, and 200 μL of complete medium was added to each well. The mixed cells were cultured in a CO2 incubator at 37 °C for 16 hours.

[0505] After co-culturing, the cell culture plate was centrifuged at 400 g for 5 minutes, 160 μL of supernatant was aspirated and removed from each well, and 50 μL of the supernatant from each well was transferred to a new 96-well flat-bottom plate (Beyotime, Cat. No. FPT019) for LDH assay. The remaining cells were cryopreserved at -80 °C for cytokine measurement. The cell pellet was subjected to flow cytometry for detection of cell death.

[0506] Detection of tumor cell death by LDH: 50 μL of cell supernatant was added to each well of a 96-well white-bottom plate, followed by 50 μL of assay solution. After incubation at room temperature in the dark for 10-30 minutes (depending on cell type and reaction color development), 50 μL of stop solution was added. Within 1 hour after adding the stop solution, absorbance was measured at 492 / 650 nm using a multifunctional microplate reader (SPARK). Wells containing tumor cells and immune cells at antibody concentration 0 were defined as the background value, wells containing only tumor cells as the minimum value, and wells containing tumor cells treated with lysis buffer as the maximum value. The virulence rate was calculated as (sample value - background value) / (maximum value - minimum value).

[0507] Detection of tumor cell death by flow cytometry: The cell culture plate described above was washed once with 200 μL of FACS buffer (1×PBS, 2% FBS, 2 mM EDTA) per well, and then centrifuged. The supernatant was removed, and 45 μL of DCM staining solution (prepared at 1:2000 with FACS buffer) was added to each well. The plate was incubated at 4 °C in the dark for 20 minutes, washed once with 200 μL of FACS buffer, and dried. The cells were resuspended in 150 μL of FACS buffer and measured by flow cytometry (BD). The motility rate was determined by whether the cells were DCM-positive and CTV-positive (DCM + CTV + ) Cell count / CTV positive (CTV + ) This was calculated as the total number of cells.

[0508] Cytokine measurement using Cisbio: A standard stock solution was obtained by dissolving the standard substance in H2O, and Std 7 (Standard 7) was prepared by diluting it 3-fold with the diluent. Subsequently, 2-fold serial dilutions were performed in 6 wells to prepare Std 1-6, and Std 0 was used as a blank control for the dilutions. 16 μL of the supernatant was added to each well of a 96-well plate (#66PL96025), and 16 μL of the standard substance was also added to each well. Eu Cryptate and d2 antibody were both diluted 1:20 with detection buffer. The two dilutions were mixed 1:1, and 4 μL of this mixture was added to each well. The plate was sealed and incubated at room temperature for 2 hours. The luminescence signal was measured using a multifunctional microplate reader (SPARK), and the readings at 665 nm / 620 nm were taken as the relative cytokine content.

[0509] Experimental results: The antibody used in the example was able to activate and mediate the dose-dependent killing effect of PBMCs on MM cells (Figures 3A-3O, Tables 12-24). 1. The results of the antibody's killing effect on H929 ko GPRC5D and H929 ko BCMA expressing a single TAA are shown in Figures 3A-3F and Tables 12-16. The specific results are as follows:

[0510] In cells expressing TAA, the example antibody dose-dependently activated T cell killing in PBMCs, similar to the corresponding antibody control molecule. In contrast, no killing effect was observed in non-target cells that do not express TAA (Figure 3P), indicating that the killing was specific and dependent on binding to the corresponding TAA. The higher the affinity of the example antibody for the anti-CD3 side, the higher the tumor cell killing ability. Specifically, molecules containing a heavy chain variable region derived from sp34.24 (SEQ ID NO: 77) in the antigen-binding site that specifically binds to CD3 (indicated as (24) in the figure) were more potent than molecules containing a heavy chain variable region derived from sp34.87 (SEQ ID NO: 78) (indicated as (87) in the figure).

[0511] Results from PBMC killing mediation against H929 ko GPRC5D showed that the example antibodies in B3, B3b, and B5 formats were superior to the F2 format molecule in mediating tumor cell killing, suggesting that the B3 / B3b / B5 design, which avoids scFv, improved BCMA functionality.

[0512] Results from PBMC killing mediation against H929 ko BCMA showed that the example antibodies in B3 and B5 formats were equivalent to the F2 format molecule and superior to the B3b format molecule.

[0513] The above results indicate that the anti-GPRC5D and anti-BCMA sides of the example antibodies in formats B3, B5, and B3b function as TCE TAA antibodies and can mediate T cell-mediated tumor cell killing.

[0514] 2. In H929 and L363 cells expressing both antigens (Figures 3G-3M, Tables 18-24), BCMA and GPRC5D expression on the surface of L363 cells was low, while BCMA and GPRC5D expression on the surface of H929 cells was high (compared to the antibodies disclosed in WO2022174813). The specific results are as follows.

[0515] In H929 cells, as described above, molecules containing a heavy chain variable region (SEQ ID NO: 77) derived from sp34.24 as the antigen-binding site that specifically binds to CD3 were more potent than molecules containing a heavy chain variable region (SEQ ID NO: 78) derived from sp34.87 as the corresponding antigen-binding site. Furthermore, the killing effect of the triplicate example antibodies B3(24) and B3-7F5.3(24) was superior to that of the JNJ-derived control bispecific antibody molecules, namely JNJ-BCMA / CD3 and JNJ-GPRC5D / CD3, and slightly inferior to or equivalent to that of the Roche-derived control bispecific antibody molecules, namely Roche-GPRC5D and Roche-BCMA / CD3.

[0516] On the other hand, in a comparison of cytokine release associated with the killing of H929 cells, the triplicate antibody used in the example did not show a significant difference in induced cytokine release compared to control bispecific antibodies such as JNJ-BCMA / CD3, JNJ-GPRC5D / CD3, and REGN-BCMA / CD3 (Figures 3Q-3R).

[0517] However, under conditions where target cells were absent, the triplicate antibody used in the example did not induce nonspecific cytokine release, similar to the control bispecific antibodies such as JNJ-BCMA / CD3, JNJ-GPRC5D / CD3, Roche-GPRC5D / CD3, Roche-BCMA / CD3, and REGN-BCMA / CD3; in other words, the level of release was equivalent to that of the hIgG control (Figure 3S-3T).

[0518] In L363 cells, the killing effect of the example trispecific antibodies B3(24) and B3-7F5.3(24) was stronger than that of the JNJ-derived control bispecific antibody molecules (JNJ-BCMA / CD3 and JNJ-GPRC5D / CD3) and equivalent to that of the Roche-derived control bispecific antibody molecules (Roche-GPRC5D / CD3 and Roche-BCMA / CD3). The anti-GPRC5D and anti-BCMA sides of the example trispecific antibodies of the present invention also showed functionality when separated from the control molecules (Inno-B, Inno-G, or Inno-G-7F5.3) and used for killing. The JNJ and Roche-derived control BCMA / CD3 and GPRC5D / CD3 bispecific antibody molecules were mixed in a 1:1 ratio and compared with the example trispecific antibodies of the present invention at the same concentration. In this comparison, the killing of L363 cells mediated by the triplicate antibody of the present invention was superior to the combined use of the control BCMA / CD3 and GPRC5D / CD3 bispecific antibody molecules. These results indicate that the anti-GPRC5D and anti-BCMA sides of the triplicate antibody have a synergistic effect in killing tumor cells, and that this is superior to the combined use of two bispecific antibodies.

[0519] 3. After mixing H929 ko GPRC5D and H929 ko BCMA expressing a single TAA with parental H929 cells expressing a dual antigen in a 1:1:1 ratio, the example triplicate antibody of the present invention showed a higher killing curve, i.e., a higher maximum killing rate, than the control molecule (Figures 3N and 3O).

[0520] Table 12: Killing effect of PBMCs mediated by example antibodies against H929 ko GPRC5D cells - 1 ("NA" indicates analysis not performed).

[0521] [Table 17]

[0522] Table 13: Killing effect of PBMCs mediated by example antibodies on H929 ko GPRC5D cells - 2.

[0523] [Table 18]

[0524] Table 14: Killing effect of PBMCs mediated by example antibodies on H929 ko GPRC5D cells -3.

[0525] [Table 19]

[0526] Table 15: Killing effect of PBMCs mediated by example antibodies against H929 ko BCMA cells - 1 ("NA" indicates analysis not performed).

[0527] [Table 20]

[0528] Table 16: Killing effect of PBMCs mediated by example antibodies against H929 ko BCMA cells - 2 ("NA" indicates analysis not performed).

[0529] [Table 21]

[0530] Table 17: Killing effect of PBMCs mediated by example antibodies against H929 ko BCMA cells - 3 ("NA" indicates analysis not performed).

[0531] [Table 22]

[0532] Table 18 Example: Antibody-mediated killing effect of PBMCs on H929 cells - 1.

[0533] [Table 23]

[0534] Table 19 Example: The killing effect of PBMCs mediated by antibodies on H929 cells - 2.

[0535] [Table 24]

[0536] Table 20 Example: Antibody-mediated killing effect of PBMCs on H929 cells - 3.

[0537] [Table 25]

[0538] Table 21 Example: Antibody-mediated killing effect of PBMCs on L363 cells - 1.

[0539] [Table 26]

[0540] Table 22 Example: Antibody-mediated killing effect of PBMCs on L363 cells - 2.

[0541] [Table 27]

[0542] Table 23 Example: Antibody-mediated killing effect of PBMCs on L363 cells - 3.

[0543] [Table 28]

[0544] Table 24 Example: Antibody-mediated killing effect of PBMCs on L363 cells - 4.

[0545] [Table 29]

[0546] Killing assay against mutant antigen-expressing cells The experimental procedure was as follows: PBMC cells (Saily, SLB-HP100B) were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and added to 9 mL of serum-free medium. The mixture was centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cells were resuspended in phenol red-free 1640 medium with 10% FBS, and the cell density was increased to 2 × 10⁶. 6 The cell density was adjusted to cells / mL. Tumor cells in the logarithmic growth phase were harvested and washed twice with PBS to remove excess serum. The BCMA mutant overexpressing GS-CHO cell lines constructed in Example 1 were centrifuged at 300 × g for 5 minutes. The supernatant was discarded and the cell pellet was retained. The cells were resuspended in phenol red-free medium and the cell density was adjusted to 4 × 10⁶. 5The solution was adjusted to cells / mL. The antibody was sequentially diluted 10-fold in complete medium to obtain a total of 8 concentrations (initial concentration 200 nM, final maximum concentration 50 nM), and a final concentration of 0 was also added. 50 μL of tumor cells, 50 μL of sequentially diluted antibody, and 100 μL of PBMC cells were added to a 96-well cell culture plate (U-bottom, COSTAR, Cat.7007) and co-cultured. Wells for spontaneous LDH release by target cells (containing only 50 μL of tumor cells) and wells for maximum LDH release by target cells (50 μL of tumor cells with lysis buffer [included in the LDH kit, diluted 1:10] added) were prepared, and 200 μL of complete medium was added to each well. The mixed cells were cultured in a CO2 incubator at 37°C for 16 hours.

[0547] After co-culturing, the culture plate was centrifuged at 400g for 5 minutes, and 160 μL of supernatant was removed from each well. Then, 50 μL of the supernatant from each well was transferred to a new 96-well flat-bottom plate (Beyotime, Cat.FPT019) and subjected to the LDH assay.

[0548] Detection of tumor cell death by LDH: 50 μL of cell supernatant was added to each well of a 96-well white-bottom plate, followed by 50 μL of assay solution. After incubation at room temperature in the dark for 10-30 minutes (depending on cell type and reaction color development), 50 μL of stop solution was added. Within 1 hour after adding the stop solution, absorbance was measured at 492 / 650 nm using a multifunctional microplate reader (SPARK). Wells containing tumor cells and immune cells at antibody concentration 0 were defined as the background value, wells containing only tumor cells as the minimum value, and wells containing tumor cells treated with lysis buffer as the maximum value. The virulence rate was calculated as (sample value - background value) / (maximum value - minimum value).

[0549] Experimental results: B3(24) dose-dependently activated and mediated the killing effect of PBMCs against GS-CHO cells overexpressing BCMA mutants (GS CHO-hBCMA-P34del, GS CHO-hBCMA-S30del, GS CHO-hBCMA-R39A, GS CHO-hBCMA-R27P). On the other hand, Teclistamab did not exhibit in vitro killing activity against BCMA mutants R27P and R39A (Figure 3U).

[0550] Example 6. Overcoming the effects of soluble BCMA (sBCMA) with a triplicate antibody. The soluble form of BCMA (sBCMA) is produced by cleavage by γ-secretase, which leads to its shedding from the plasma cell surface. It is elevated in MM patients and associated with poor clinical outcomes (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5395119 / ). sBCMA consists of the extracellular domain and part of the transmembrane domain of BCMA and functions as a soluble decoy that not only reduces the density of target antigens but can also limit the efficacy of the BCMA / CD3 molecule. The GPRC5D / BCMA / CD3 molecule has a GPRC5D end that is not restricted by sBCMA and is therefore theoretically less affected by sBCMA.

[0551] The experimental procedure was as follows: Human cryopreserved PBMC cells (SAILY) were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and added to 9 mL of serum-free medium. The mixture was centrifuged at 300 g for 5 minutes, and the supernatant was discarded. The cells were resuspended in 1% FBS-supplemented phenol red-free 1640 medium (complete medium), and the cell density was increased to 4 × 10⁶. 6 The cell culture medium was adjusted to cells / mL. The cell culture medium was collected in a 50 mL sterile centrifuge tube and centrifuged at 300 × g for 5 minutes. The supernatant was discarded, and the cell pellet was retained. The cell concentration was adjusted to 4 × 10⁶ in complete medium. 5The antibody was adjusted to the required cell / mL concentration. The antibody was serially diluted 10-fold in complete medium to a concentration of 200 nM. Simultaneously, human recombinant BCMA (hBCMA; stock solution 0.5 mg / mL, prepared in PBS) was diluted to 800 ng / mL in complete medium to obtain sBCMA solution. 50 μL of tumor cells (H929), 50 μL of serially diluted antibody, 50 μL of PBMC cells, and 50 μL of sBCMA solution were added to a 96-well cell culture plate (U-bottom type, Costar), and at this point the sBCMA concentration in the co-culture system was 200 ng / mL. The mixture was cultured at 37°C in a CO2 incubator for 16 hours. After co-culture, the culture plate was centrifuged at 400 g for 5 minutes, and 50 μL of the supernatant from each well was transferred to a new 96-well flat-bottom plate (Beyotime, Cat. No. FPT019) and subjected to the LDH assay. For the cell pellet, the percentage of CD69-positive cells among T cells was measured by flow cytometry (BD). See Example 5 for information on the cells used.

[0552] Detection of tumor cell death by LDH: 50 μL of cell supernatant was added to each well of a 96-well white-bottom plate, followed by 50 μL of assay solution. After incubation at room temperature in the dark for 10-30 minutes (depending on cell type and reaction color development), 50 μL of stop solution was added. Within 1 hour after adding the stop solution, absorbance was measured at 492 / 650 nm using a multifunctional microplate reader (SPARK). Wells containing tumor cells and immune cells at antibody concentration 0 were defined as the background value, wells containing only tumor cells as the minimum value, and wells containing tumor cells treated with lysis buffer as the maximum value. The virulence rate was calculated as (sample value - background value) / (maximum value - minimum value).

[0553] Detection of CD8 and CD4 T cell activation by flow cytometry: The cell culture plate described above was washed once with 200 μL of FACS buffer (1×PBS, 2% FBS, 2 mM EDTA) per well, and then centrifuged. The supernatant was removed, and 45 μL of staining solution (prepared with FACS buffer) was added to each well. The plate was incubated in the dark at 4 °C for 20 minutes, washed once with 200 μL of FACS buffer, and dried. The cells were resuspended in 150 μL of FACS buffer and measured using a flow cytometer. The percentage of CD69-positive cells in CD8 T cells and CD4 T cells was calculated.

[0554] Reagents and materials:

[0555] [Table 30]

[0556] The experimental results are shown in Figures 4A, 4B, and 4C, and in Tables 25 and 26. The results indicate that T cell activation and H929 cell killing mediated by the trispecific antibody are less affected by sBCMA.

[0557] Table 25 Example: Activation of T cells mediated in PBMCs by antibody (w / o: none, w: present)

[0558] [Table 31]

[0559] Table 26 Example: Activation of T cells mediated in PBMCs by antibody (w / o: none, w: present)

[0560] [Table 32]

[0561] Example 7. Antitumor efficacy of a triplicate antibody in a humanized mouse tumor model. Female NOG mice (35-48 days old) were purchased from Beijing Vitalstar Biotechnology Co., Ltd. and are SPF mice. After 5-7 days of acclimatization and quarantine upon arrival, testing was initiated.

[0562] Tumor cells were subcultured using a standard method for subsequent in vivo testing. Cells were harvested by centrifugation and dispersed in PBS. PBMC cells were administered to NOG mice in 4 × 10⁶ units. 6 Cells were administered intravenously at a dose of 200 μL per mouse. Three days after PBMC cell inoculation, the backs and right sides of the abdomen of the mice were shaved, and tumor cells were subcutaneously administered at a dose of 200 μL per mouse (inoculation density: H929 ko BCMA: 4 × 10). 6 Cells / animal, H929 ko GPRC5D: 4×10 6 Cells / mouse, MM.1S (MM cells, Nanjing Cobioer, CBP60239): 3×10 6 / fish, L363:1×10 6 (Cells / mouse). On day 7 after tumor cell inoculation, mice were divided into groups (6-7 mice per group) based on tumor volume, and intraperitoneal administration was performed (see Figure 5 and brief descriptions of the drawings for details on administration frequency and dose). Tumor volume and body weight of mice were monitored twice a week. Tumor volume measurement: The maximum length of the longest diameter (L) and shortest diameter (W) of the tumor was measured using calipers, and the tumor volume was calculated using the following formula: V = L × W² / 2. Mouse body weight was measured using an electronic balance. Throughout the study period, mice were euthanized if the tumor reached the endpoint or if body weight loss exceeded 20%. Tumor sizes were tallied.

[0563] Experimental results: H929 ko GPRC5D tumor (Figure 5A): The B3(24) molecule effectively inhibited the proliferation of H929 ko GPRC5D cells, and its antitumor activity was generally equivalent to that of the JNJ-BCMA / CD3 control molecule, and more potent than the B3(87), REGN-BCMA / CD3, and F2(87) molecules.

[0564] H929 ko BCMA tumors (Figure 5B): The B3(24) molecule effectively inhibited the proliferation of H929 ko BCMA cells, and its antitumor activity was generally equivalent to that of the JNJ-GPRC5D / CD3 control molecule. It was more potent than all BCMA / CD3 combinations and (JNJ-GPRC5D / CD3 + JNJ-BCMA / CD3), but slightly weaker than Roche-GPRC5D / CD3 and (Roche-BCMA / CD3 + Roche-GPRC5D / CD3) combinations.

[0565] MM1S tumor (Figure 5C): Both the antibody used in the example and the control molecule showed good antitumor effects.

[0566] L363 tumor (Figure 5D): The B3(24) molecule effectively suppressed the proliferation of L363 cells, and its antitumor activity was more potent than that of JNJ-GPRC5D / CD3, JNJ-BCMA / CD3, and (JNJ-BCMA / CD3 + JNJ-GPRC5D / CD3) combinations, and close to that of Roche-GPRC5D / CD3, Roche-BCMA / CD3, and (Roche-GPRC5D / CD3 + Roche-GPRC5D / CD3) combinations.

[0567] The B3(24) molecule did not show any difference in its effect on mouse body weight compared to other control molecules in any of the models.

[0568] [Table 33-1]

[0569] [Table 33-2]

[0570] [Table 33-3]

[0571] [Table 33-4]

[0572] Table 34-1

[0573] Table 34-2

[0574] Table 35-1

[0575] Table 35-2

[0576] Table 36-1

[0577] Table 36-2

[0578] Table 36-3

[0579] Table 36-4

[0580] Table 36-5

[0581] Table 36-6

[0582] Table 36-7

[0583] Table 36-8

[0584] Table 36-9

[0585] Table 36-10

[0586] Table 36-11

[0587] Table 36-12

[0588] Table 36-13

[0589] Table 36-14

[0590] Table 36-15

[0591] Table 36-16

[0592] Table 36-17

Claims

1. A triplicate antibody that specifically binds to BCMA, comprising a first antigen-binding region that specifically binds to BCMA, and second and third antigen-binding regions that specifically bind to other antigens.

2. The triplicate antibody according to claim 1, wherein the second antigen-binding region specifically binds to CD3 and / or the third antigen-binding region specifically binds to GPRC5D.

3. The triplicate antibody according to claim 1 or 2, wherein the first antigen-binding region, the second antigen-binding region, and the third antigen-binding region are, respectively, a first Fab, a second Fab, and a third Fab.

4. A triplicate antibody according to claim 3, wherein, (1) The first Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, and the third Fab fuses with the N-terminus of VH of the Fab heavy chain of the second Fab fragment at the C-terminus of CH1 of the Fab heavy chain; or The third Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, and the first Fab fuses with the N-terminus of VH of the Fab heavy chain of the second Fab fragment at the C-terminus of CH1 of the Fab heavy chain; or (2) The first Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, and the third Fab fuses with the C-terminus of the Fc region fused to the second Fab at the N-terminus of VH of the Fab heavy chain; or The third Fab fuses with one CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, the second Fab fuses with another CH2 or hinge region of the Fc region of the Fc heterodimer at the C-terminus of CH1 of the Fab heavy chain, and the first Fab fuses with the C-terminus of the Fc region fused to the second Fab at the N-terminus of VH of the Fab heavy chain; Trispecific antibodies.

5. The triplicate antibody according to claim 4, wherein the fusion includes direct fusion or linker-mediated fusion.

6. A triplicate antibody according to claim 5, characterized in that the linker is (GGGGS)n and n = 1, 2, 3, or 4.

7. The triplicate antibody according to any one of claims 3 to 6, wherein the second Fab comprises a charge mutation, and the first or third Fab fused to the second Fab, or fused to the second Fab via the Fc region, comprises a charge mutation and a disulfide bond remodeling mutation.

8. A triplicate antibody according to claim 7, wherein the charge mutation is a mutation to D or K at position 39 of VH of Fab, and a mutation to K or D at position 38 of VL of Fab, for example, the second Fab comprises VH containing 39D and VL containing 38K, and the first or third Fab fused to the second Fab via an Fc region comprises VH containing 39K and VL containing 38D; or the second Fab comprises VH containing 39K and VL containing 38D, and the first or third Fab fused to the second Fab via an Fc region comprises VH containing 39D and VL containing 38K.

9. The triplicate antibody according to claim 7 or 8, wherein the disulfide bond remodeling mutation comprises F126C in CH1 of Fab and Q124C in CL of Fab.

10. The Fab containing the disulfide bond remodeling mutation includes CH1, and the CH1 (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 85, and containing F126C, or (ii) The triplicate antibody according to claim 9, comprising or comprising the amino acid sequence shown in SEQ ID NO:

85.

11. The Fab containing the disulfide bond remodeling mutation includes CL, and the CL is (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 97, and containing Q124C, or (ii) The triplicate antibody according to claim 9 or 10, comprising or consisting of the amino acid sequence shown in SEQ ID NO:

97.

12. The triplicate antibody according to claim 9, wherein the Fab containing a disulfide bond remodeling mutation comprises CH1 and CL, wherein CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 85, and CL comprises or consists of the amino acid sequence shown in SEQ ID NO:

97.

13. The triplicate antibody according to claim 7 or 8, wherein the disulfide bond remodeling mutations include F126C and C220S in CH1 of Fab, and Q124C and C214S in CL of Fab.

14. The Fab containing the disulfide bond remodeling mutation includes CH1, and the CH1 (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 86, and containing F126C and C220S, or The triplicate antibody according to claim 13, wherein (ii) comprises or consists of the amino acid sequence shown in SEQ ID NO:

86.

15. The Fab containing the disulfide bond remodeling mutation includes CL, and the CL is (i) an amino acid sequence having at least 90% identity with the amino acid sequence shown in Sequence ID No. 98, and containing Q124C and C214S, or (ii) The triplicate antibody according to claim 13 or 14, wherein CL comprises or consists of the amino acid sequence shown in SEQ ID NO:

98.

16. The triplicate antibody according to claim 13, wherein the Fab containing a disulfide bond remodeling mutation comprises CH1 and CL, wherein CH1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 86, and CL comprises or consists of the amino acid sequence shown in SEQ ID NO:

98.

17. The triplicate antibody according to any one of claims 1 to 16, wherein the triplicate antibody comprises a first Fc region and a second Fc region, and the first Fc region and the second Fc region may be the same or different.

18. The triplicate antibody according to claim 17, wherein the first Fc region and the second Fc region are human IgG Fc such as human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc, and include or consist of the amino acid sequence shown in SEQ ID NO: 87 or 88, or include an amino acid sequence having at least 90%, for example 95%, 96%, 97%, or 99% or more identity with said amino acid sequence.

19. The triplicate antibody according to claim 18, wherein the first Fc region and / or the second Fc region comprises the L234A / L235A mutation.

20. The triplicate antibody according to claim 19, wherein the first Fc region and / or the second Fc region comprises the amino acid sequence shown in SEQ ID NO: 89 or 90, or comprises an amino acid sequence having at least 90%, for example 95%, 96%, 97%, or 99% or more identity with the amino acid sequence shown in SEQ ID NO: 89 or 90, and comprises the L234A / L235A amino acid substitution.

21. A trispecific antibody according to any one of claims 17 to 20, wherein one of the first Fc region and the second Fc region contains a Knob mutation and the other contains a Hole mutation.

22. The triplicate antibody according to claim 21, wherein one Fc region contains the amino acid substitution T366W, and the other Fc region contains the amino acid substitutions T366S, L368A, and Y407V (numbering according to the EU index).

23. The triplicate antibody according to claim 22, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 91, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

92.

24. The triplicate antibody according to claim 23, wherein one Fc region polypeptide contains or comprises the amino acid sequence shown in SEQ ID NO: 91, and the other Fc region polypeptide contains or comprises the amino acid sequence shown in SEQ ID NO:

92.

25. The triplicate antibody according to claim 17, wherein one Fc region comprises the amino acid substitutions L234A / L235A and T366W, and the other Fc region comprises the amino acid substitutions L234A / L235A, T366S, L368A and Y407V (numbering follows the EU index).

26. The triplicate antibody according to claim 25, wherein one Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 94, and the other Fc region polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

93.

27. The triplicate antibody according to claim 26, wherein one Fc region polypeptide contains or comprises the amino acid sequence shown in SEQ ID NO: 94, and the other Fc region polypeptide contains or comprises the amino acid sequence shown in SEQ ID NO:

93.

28. A trispecific antibody according to any one of claims 1 to 27, wherein the first antigen-binding region that specifically binds to BCMA comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein A triple-specific antibody comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 57, or comprising; HCDR2 containing the amino acid sequence shown in SEQ ID NO: 58, or comprising; HCDR3 containing the amino acid sequence shown in SEQ ID NO: 59, or comprising; LCDR1 containing the amino acid sequence shown in SEQ ID NO: 61, or comprising; LCDR2 containing the amino acid sequence shown in SEQ ID NO: 62, or comprising; and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 63, or comprising.

29. The triplicate antibody according to claim 28, wherein the VH of the first antigen-binding region that specifically binds to BCMA includes or comprises the amino acid sequence shown in SEQ ID NO: 56, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence.

30. The triplicate antibody according to claim 28 or 29, wherein the VL of the first antigen-binding region that specifically binds to BCMA includes or comprises the amino acid sequence shown in SEQ ID NO: 60, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence.

31. The triplicate antibody according to claim 30, wherein the first antigen-binding region that specifically binds to BCMA comprises VH and VL, the VH having the amino acid sequence shown in SEQ ID NO: 56, and the VL of the first antigen-binding region that specifically binds to BCMA having the amino acid sequence shown in SEQ ID NO:

60.

32. The first antigen-binding region that specifically binds to BCMA comprises VH and VL, wherein VH comprises 39K and VL comprises 38D, or VH comprises 39D and VL comprises 38K, for example, the VH of the first antigen-binding region that specifically binds to BCMA is (i) an amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 76, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 76, Furthermore, it includes the Q39K mutation, and / or the VL of the first antigen-binding region comprises or consists of the amino acid sequence shown in SEQ ID NO: 75, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 75, and comprises the Q38D mutation. The triplicate antibody according to claim 28.

33. A triplicate antibody according to any one of claims 1 to 32, wherein the third antigen-binding region specifically binds to GPRC5D, and comprises a heavy chain variable region VH and a light chain variable region VL, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, where, The HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 41, or consists of; the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 42, or consists of; the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 43, or consists of; the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 45, or consists of; the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 46, or consists of; the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 47, or consists of; or The HCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 49; the HCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 50; the HCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 51; the LCDR1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 53; the LCDR2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 54; the LCDR3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 55; Trispecific antibodies.

34. The VH of the third antigen-binding region that specifically binds to GPRC5D, (i) an amino acid sequence comprising or consisting of the amino acid sequence shown in Sequence ID No. 40, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No. 40, (ii) The trispecific antibody according to claim 33, comprising or consisting of the amino acid sequence shown in Sequence ID No. 48, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No.

48.

35. The VL of the third antigen-binding region that specifically binds to GPRC5D, (i) an amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO: 44, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 44, (ii) A trispecific antibody according to claim 33 or 34, comprising or consisting of the amino acid sequence shown in SEQ ID NO: 52, or comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

52.

36. The triple-specific antibody according to claim 35, wherein the third antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein VH consists of the amino acid sequence shown in SEQ ID NO: 48 and VL consists of the amino acid sequence shown in SEQ ID NO: 52, or VH consists of the amino acid sequence shown in SEQ ID NO: 40 and VL consists of the amino acid sequence shown in SEQ ID NO:

44.

37. The third antigen-binding region that specifically binds to GPRC5D comprises VH and VL, wherein VH comprises 39K and VL comprises 38D, or VH comprises 39D and VL comprises 38K, for example, The VH of the third antigen-binding region that specifically binds to GPRC5D (i) containing or consisting of the amino acid sequence shown in Sequence ID No. 80, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No. 80, Furthermore, it includes the Q39K mutation, Furthermore, the VL of the third antigen-binding region that specifically binds to GPRC5D contains or consists of the amino acid sequence shown in SEQ ID NO: 79, or contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 79, and contains the Q38D mutation. A triplicate antibody according to any one of claims 33 to 36.

38. A triplicate antibody according to any one of claims 1 to 37, wherein the second antigen-binding site specifically binds to CD3 and comprises a VH (heavy chain variable region) and a VL (light chain variable region), the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3, where, A triple-specific antibody comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 65, or comprising; HCDR2 containing the amino acid sequence shown in SEQ ID NO: 66, or comprising; HCDR3 containing the amino acid sequence shown in SEQ ID NO: 67 or 73, or comprising; LCDR1 containing the amino acid sequence shown in SEQ ID NO: 69, or comprising; LCDR2 containing the amino acid sequence shown in SEQ ID NO: 70, or comprising; and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 71, or comprising.

39. The triplicate antibody according to claim 38, wherein the VH of the second antigen-binding region that specifically binds to CD3 includes or comprises the amino acid sequence shown in SEQ ID NO: 64 or 72, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence.

40. The triplicate antibody according to claim 38 or 39, wherein the VL of the second antigen-binding region that specifically binds to CD3 includes or comprises the amino acid sequence shown in SEQ ID NO: 68, or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with said amino acid sequence.

41. The triplicate antibody according to claim 40, wherein the second antigen-binding region that specifically binds to CD3 comprises VH and VL, the VH consists of the amino acid sequence shown in SEQ ID NO: 64 or 72, and the VL of the second antigen-binding region consists of the amino acid sequence shown in SEQ ID NO:

68.

42. A trispecific antibody according to any one of claims 38 to 41, wherein the second antigen-binding region that specifically binds to CD3 comprises VH and VL, wherein VH comprises 39K and VL comprises 38D, or VH comprises 39D and VL comprises 38K.

43. The VH of the second antigen-binding region that specifically binds to CD3, (i) containing or consisting of the amino acid sequence shown in Sequence ID No. 77 or 78, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No. 77 or 78, and containing the Q39D mutation, Furthermore, the VL of the second antigen-binding region that specifically binds to CD3 (i) containing or consisting of the amino acid sequence shown in Sequence ID No. 74, (ii) an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in Sequence ID No. 74, and containing the Q38K mutation, The triplicate antibody according to claim 42.

44. A triplicate antibody according to any one of claims 3 to 43, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer, consisting of five polypeptide chains, and further... Peptide chain 1# containing the Fab heavy chain and Fc domain that specifically bind to GPRC5D in this order, Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain 3# containing a Fab heavy chain that specifically binds to BCMA, a Fab heavy chain that specifically binds to CD3, and an Fc domain in this order, Peptide chain 4# containing a Fab light chain that specifically binds to BCMA, Peptide chain 5# containing a Fab light chain that specifically binds to CD3 It consists of peptide chains, Here, the Fab heavy chain of peptide chain 1# is paired with the Fab light chain of peptide chain 2# to form the 3rd Fab, and the two Fab heavy chains of peptide chain 3# are paired with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the 1st Fab and the 2nd Fab, respectively, making it a triple-specific antibody.

45. A triplicate antibody according to claim 44, wherein, The peptide chain 1# contains the amino acid sequence shown in SEQ ID NO: 5, or contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5, or consists of the amino acid sequence shown in SEQ ID NO:

5. The peptide chain 2# contains the amino acid sequence shown in SEQ ID NO: 4, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4, or consists of the amino acid sequence shown in SEQ ID NO:

5. The peptide chain 3# contains the amino acid sequence shown in SEQ ID NO: 3 or 6, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3 or 6, or consists of the amino acid sequence shown in SEQ ID NO: 3 or 6. The peptide chain 4# contains the amino acid sequence shown in SEQ ID NO: 2, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2, or consists of the amino acid sequence shown in SEQ ID NO: 2, and / or The peptide chain 5# is a triple-specific antibody comprising an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that consists of the amino acid sequence shown in SEQ ID NO:

1.

46. A triplicate antibody according to claim 44, wherein, The peptide chain 1# contains the amino acid sequence shown in SEQ ID NO: 15, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 15, or consists of the amino acid sequence shown in SEQ ID NO:

15. The peptide chain 2# contains the amino acid sequence shown in SEQ ID NO: 14, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 14, or consists of the amino acid sequence shown in SEQ ID NO:

14. The peptide chain 3# contains the amino acid sequence shown in SEQ ID NO: 3 or 6, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 3 or 6, or consists of the amino acid sequence shown in SEQ ID NO: 3 or 6. The peptide chain 4# contains the amino acid sequence shown in SEQ ID NO: 2, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2, or consists of the amino acid sequence shown in SEQ ID NO: 2, and / or The peptide chain 5# is a triple-specific antibody comprising an amino acid sequence that includes the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence that consists of the amino acid sequence shown in SEQ ID NO:

1.

47. A triplicate antibody according to any one of claims 1 to 43, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer and consists of five polypeptide chains. Peptide chain 1# containing the Fab heavy chain and Fc domain that specifically bind to BCMA in this order, Peptide chain 2# containing a Fab heavy chain that specifically binds to BCMA, Peptide chain 3# containing a Fab heavy chain that specifically binds to GPRC5D, a Fab heavy chain that specifically binds to CD3, and an Fc domain in this order, Peptide chain 4# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain 5# containing a Fab light chain that specifically binds to CD3 It consists of peptide chains, Here, the Fab heavy chain of peptide chain 1# is paired with the Fab light chain of peptide chain 2# to form the first Fab, and the two Fab heavy chains of peptide chain 3# are paired with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the third Fab and the second Fab, respectively, making it a triple-specific antibody.

48. A triplicate antibody according to claim 47, wherein, The peptide chain 1# contains the amino acid sequence shown in SEQ ID NO: 11, or contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 11, or consists of the amino acid sequence shown in SEQ ID NO:

11. The peptide chain 2# contains the amino acid sequence shown in SEQ ID NO: 10, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10, or consists of the amino acid sequence shown in SEQ ID NO:

10. The peptide chain 3# contains the amino acid sequence shown in SEQ ID NO: 9, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 9, or consists of the amino acid sequence shown in SEQ ID NO:

9. Peptide chain 4# contains the amino acid sequence shown in SEQ ID NO: 8, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 8, or consists of the amino acid sequence shown in SEQ ID NO: 8, and / or Peptide chain 5# is a triple-specific antibody comprising an amino acid sequence containing the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1, or consisting of the amino acid sequence shown in SEQ ID NO:

1.

49. A triplicate antibody according to any one of claims 1 to 43, wherein the triplicate antibody is a left-right asymmetric IgG-like pentamer and consists of five polypeptide chains. Peptide chain 1# containing the Fab heavy chain and Fc domain that specifically bind to GPRC5D in this order, Peptide chain 2# containing a Fab light chain that specifically binds to GPRC5D, Peptide chain 3# contains, in this order, a Fab heavy chain that specifically binds to CD3, an Fc domain, and a Fab heavy chain of an antigen-binding region that specifically binds to BCMA. Peptide chain 4# containing a Fab light chain that specifically binds to CD3, Peptide chain 5# containing the Fab light chain, which is an antigen-binding site that specifically binds to BCMA. It consists of peptide chains, Herein, the Fab heavy chain of peptide chain 1# is paired with the Fab light chain of peptide chain 2# to form the 3rd Fab, and the two Fab heavy chains of peptide chain 3# are paired with the Fab light chains of peptide chain 4# and peptide chain 5#, respectively, to form the 2nd Fab and the 1st Fab, respectively, making it a triple-specific antibody.

50. A triplicate antibody according to claim 49, wherein, The peptide chain 1# contains the amino acid sequence shown in SEQ ID NO: 5, or contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 5, or consists of the amino acid sequence shown in SEQ ID NO:

5. The peptide chain 2# contains the amino acid sequence shown in SEQ ID NO: 4, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 4, or consists of the amino acid sequence shown in SEQ ID NO:

4. The peptide chain 3# contains the amino acid sequence shown in SEQ ID NO: 7, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 7, or consists of the amino acid sequence shown in SEQ ID NO:

7. The peptide chain 4# contains the amino acid sequence shown in SEQ ID NO: 1, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 1, or consists of the amino acid sequence shown in SEQ ID NO: 1, and / or A triplicate antibody wherein peptide chain 5# contains the amino acid sequence shown in SEQ ID NO: 2, or contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 2, or consists of the amino acid sequence shown in SEQ ID NO:

2.

51. A nucleic acid molecule encoding any of the chains of any of the triplicate antibodies according to any one of claims 1 to 50, or a nucleic acid molecule consisting of a nucleic acid sequence.

52. An expression vector comprising a nucleic acid molecule as described in claim 51, wherein the expression vector is, for example, a pCNDA vector (e.g., a pcDNA3.1 expression vector).

53. A host cell comprising the nucleic acid molecule described in claim 51 or the expression vector described in claim 52, wherein the host cell is preferably a prokaryotic cell or a eukaryotic cell, for example, a 293 cell or a CHO cell (e.g., a HEK293 cell).

54. A method for producing a triplicate antibody according to any one of claims 1 to 50, the method comprising culturing a host cell containing the nucleic acid molecule according to claim 51 or the expression vector according to claim 52 under conditions suitable for expressing the chain of the antibody, and optionally recovering the antibody from the host cell (or the culture medium of the host cell).

55. An immunoconjugate comprising a triplicate antibody according to any one of claims 1 to 50.

56. A pharmaceutical composition, pharmaceutical, or preparation comprising a trispecific antibody according to any one of claims 1 to 50 or an immunoconjugate according to claim 55, and optionally a pharmaceutical auxiliary material.

57. A pharmaceutical combination comprising a triplicate antibody according to any one of claims 1 to 50 or an immune conjugate according to claim 55, and one or more additional therapeutic agents (e.g., chemotherapeutic agents).

58. A method for preventing or treating cancer in a subject, comprising administering to the subject an effective amount of a trispecific antibody according to any one of claims 1 to 50, an immune conjugate according to claim 55, a pharmaceutical composition or preparation according to claim 56, or a pharmaceutical combination according to claim 57.

59. The method according to claim 58, wherein the cancer is a GPRC5D-positive cancer, a BCMA-positive cancer, a GPRC5D / BCMA-double-positive cancer, or a cancer with dual low expression of BCMA and GPRC5D, for example, a cancer with low expression of GPRC5D and / or low expression of BCMA (e.g., a cancer that has recurred after anti-BCMA therapy or anti-GPRC5D therapy).

60. A method according to claim 58 or 59, wherein the cancer is a solid tumor or a hematological malignancy, for example, multiple myeloma.

61. A method according to any one of claims 58 to 60, the method further comprising the step of administering a therapeutic means (e.g., surgical therapy or radiotherapy) and / or an additional therapeutic agent (e.g., a chemotherapeutic agent) in combination.

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